Neisseria meningitidis compositions and methods thereof

ABSTRACT

In one aspect, the invention relates to a composition including a factor H binding protein (fHBP) and a  Neisseria meningitidis  non-serogroup B capsular polysaccharide. The invention further relates to uses of a composition that includes fHBP, such as, for example, uses to elicit an immune response against  N. meningitidis  serogroup B strains and non-serogroup B strains. The compositions and methods described herein are directed to administration in humans, including adults, adolescents, toddlers, and infants.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of Ser. No. 16/704,701, filed onDec. 5, 2019, now allowed, which is a continuation of and claims thebenefit of U.S. patent application Ser. No. 16/196,150, filed on Nov.20, 2018 (now U.S. Pat. No. 10,543,267), which claims the benefit ofU.S. patent application Ser. No. 15/883,334, filed on Jan. 30, 2018 (nowU.S. Pat. No. 10,183,070), which claims the benefit of U.S. ProvisionalPatent Application 62/452,963, filed on Jan. 31, 2017, U.S. ProvisionalPatent Application 62/503,295, filed on May 8, 2017, U.S. ProvisionalPatent Application 62/613,945, filed on Jan. 5, 2018, and U.S.Provisional Patent Application No. 62/623,233, filed on Jan. 29, 2018.All of the foregoing applications are hereby incorporated by referencein their entireties.

FIELD OF THE INVENTION

The present invention relates to Neisseria meningitidis compositions andmethods thereof.

BACKGROUND OF THE INVENTION

Neisseria meningitidis is a Gram-negative encapsulated bacterium thatcan cause sepsis, meningitis, and death. N. meningitidis can beclassified into at least 12 serogroups (including serogroups A, B, C,29E, H, I, K, L, W-135 (mostly now referred to as W), X, Y and Z) basedon chemically and antigenically distinctive polysaccharide capsules.Strains with five of the serogroups (A, B, C, Y, and W135) areresponsible for the majority of disease.

Meningococcal meningitis is a devastating disease that can kill childrenand young adults within hours despite the availability of antibiotics.There is a need for improved immunogenic compositions againstmeningococcal serogroups A, B, C, Y, and W135 and/or X.

Currently, a cross-protective vaccine or composition effective against awide range of MnB and meningococcal serogroups A, C, Y, and W and/or Xisolates is not yet commercially available. For example, publishedresults-to-date relating to a licensed multi-component composition forprotection against serogroup B disease has not demonstrated a directbactericidal immune response against multiple meningococcal strains thatexpressnon-serogroup B capsular polysaccharides, at least inadolescents. Accordingly, a cross-protective vaccine or compositioneffective against diverse MnB and meningococcal serogroups A, C, Y, andW and/or X isolates is needed as is determining real-world vaccinecoverage against a panel of diverse or heterologous meningococcalstrains (e.g., representing different geographical regions).

It is a further object of the invention to provide improved schedulesfor administering a meningococcal vaccine, in particular to children.While incidence rates of invasive meningococcal disease (IMD) vary withage, incidence is often highest during infancy from age 1 month to 1year, with a second peak in incidence during adolescence. In the UnitedStates, during 1998 to 2007, the overall rate of meningococcal diseasein infants aged less than 2 years was 3.9 per 100,000. In children aged2 to 10 years, the incidence was 0.68 per 100,000, with 41% of cases inthis age group occurring in children aged 2 to 3 years. Nationalsurveillance data from Australia show the peak incidence of disease inchildren aged 4 years or less, with a secondary peak in adolescents andyoung adults; approximately 85% of all cases are attributed to serogroupB disease.

SUMMARY OF THE INVENTION

To meet these and other needs, the present invention relates toNeisseria meningitidis compositions and methods thereof.

The inventors surprisingly discovered a composition including at leastone factor H binding protein (fHBP) and at least one N. meningitidiscapsular saccharide conjugate. The composition is surprisingly stableand elicited an immune response against strains that express fHBPvariants that are homologous to the fHBP variant in the multi-componentcomposition and an immune response against strains that express fHBPvariants that are heterologous to the fHBP variant in themulti-component composition.

The composition includes a first lipidated polypeptide including theamino acid sequence set forth in SEQ ID NO: 1; a second lipidatedpolypeptide including the amino acid sequence set forth in SEQ ID NO: 2;a Neisseria meningitidis serogroup A (MenA) capsular saccharideconjugated to tetanus toxoid carrier protein (TT); a Neisseriameningitidis serogroup C (MenC) capsular saccharide conjugated totetanus toxoid carrier protein (TT); a Neisseria meningitidis serogroupW135 (MenW) capsular saccharide conjugated to tetanus toxoid carrierprotein (TT); and a Neisseria meningitidis serogroup Y (MenY) capsularsaccharide conjugated to tetanus toxoid carrier protein (TT).

In one embodiment, the composition includes a Neisseria meningitidisserogroup A (MenA) capsular saccharide conjugated to an adipic aciddihydrazide (ADH) linker by 1-cyano-4-dimethylamino pyridiniumtetrafluoroborate chemistry, wherein the linker is conjugated to tetanustoxoid carrier protein (TT) by carbodiimide chemistry (MenA_(AH)-TTconjugate); a Neisseria meningitidis serogroup C (MenC) capsularsaccharide conjugated to an ADH linker by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry, wherein the linker is conjugatedto tetanus toxoid carrier protein (TT) by carbodiimide chemistry(MenC_(AH)-TT conjugate); a Neisseria meningitidis serogroup W135 (MenW)capsular saccharide directly conjugated to tetanus toxoid carrierprotein (TT) by 1-cyano-4-dimethylamino pyridinium tetrafluoroboratechemistry, in the absence of a linker (MenW-TT conjugate); and aNeisseria meningitidis serogroup Y (MenY) capsular saccharide directlyconjugated to tetanus toxoid carrier protein (TT) by1-cyano-4-dimethylamino pyridinium tetrafluoroborate chemistry, in theabsence of a linker (MenY-TT conjugate).

In one aspect, the invention relates to a kit including (a) a firstcomposition including a lipidated MenB rLP2086 subfamily A polypeptideand a lipidated MenB rLP2086 subfamily B polypeptide; and (b) a secondcomposition including a Neisseria meningitidis serogroup A (MenA)capsular saccharide conjugated to tetanus toxoid carrier protein (TT); aNeisseria meningitidis serogroup C (MenC) capsular saccharide conjugatedto tetanus toxoid carrier protein (TT); a Neisseria meningitidisserogroup W135 (MenW) capsular saccharide conjugated to tetanus toxoidcarrier protein (TT); and a Neisseria meningitidis serogroup Y (MenY)capsular saccharide conjugated to tetanus toxoid carrier protein (TT).In one embodiment, the first composition is a liquid composition and thesecond composition is a lyophilized composition. In another embodiment,the kit does not further include any one of the following immunogeniccompositions: MENACTRA®, MENVEO®, ADACEL®, HAVRIX®, GARDASIL®, REPEVAX,or any combination thereof. In one embodiment, the kit includes any oneof ibuprofen, paracetamol, and amoxicillin.

In one aspect, the invention relates to an immunogenic compositionincluding a liquid composition including (i) a first lipidatedpolypeptide including the amino acid sequence set forth in SEQ ID NO: 1;and (ii) a second lipidated polypeptide including the amino acidsequence set forth in SEQ ID NO: 2; and a lyophilized compositionincluding a Neisseria meningitidis serogroup A (MenA) capsularsaccharide conjugated to an adipic acid dihydrazide (ADH) linker by1-cyano-4-dimethylamino pyridinium tetrafluoroborate chemistry, whereinthe linker is conjugated to tetanus toxoid carrier protein (TT) bycarbodiimide chemistry (MenA_(AH)-TT conjugate); a Neisseriameningitidis serogroup C (MenC) capsular saccharide conjugated to an ADHlinker by 1-cyano-4-dimethylamino pyridinium tetrafluoroboratechemistry, wherein the linker is conjugated to tetanus toxoid carrierprotein (TT) by carbodiimide chemistry (MenC_(AH)-TT conjugate); aNeisseria meningitidis serogroup W₁₃₅ (MenW) capsular saccharidedirectly conjugated to tetanus toxoid carrier protein (TT) by1-cyano-4-dimethylamino pyridinium tetrafluoroborate chemistry, in theabsence of a linker (MenW-TT conjugate); and a Neisseria meningitidisserogroup Y (MenY) capsular saccharide directly conjugated to tetanustoxoid carrier protein (TT) by 1-cyano-4-dimethylamino pyridiniumtetrafluoroborate chemistry, in the absence of a linker (MenY-TTconjugate). In one embodiment, the lyophilized composition isreconstituted with the liquid composition.

In another aspect, the invention relates to a method of inducing abactericidal immune response against a Neisseria meningitidis serogroupX strain. In some embodiments, the method includes administering to thehuman a composition including a fHBP protein. In some embodiments, themethod includes administering to the human a composition comprising apolypeptide comprising an amino acid sequence having at least 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or99.9% identity to the amino acid sequence set forth in any one of SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18,SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO:23, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ IDNO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39,SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO:44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ IDNO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58,SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62. In someembodiments, the method includes administering to the human acomposition including a first lipidated polypeptide including the aminoacid sequence set forth in SEQ ID NO: 1; a second lipidated polypeptideincluding the amino acid sequence set forth in SEQ ID NO: 2.

In another aspect, the invention relates to a method of inducing abactericidal immune response against a Neisseria meningitidis serogroupX strain. The method includes administering to the human a compositionthat includes a first lipidated polypeptide including the amino acidsequence set forth in SEQ ID NO: 1; a second lipidated polypeptideincluding the amino acid sequence set forth in SEQ ID NO: 2; a Neisseriameningitidis serogroup A (MenA) capsular saccharide conjugated totetanus toxoid carrier protein (TT); a Neisseria meningitidis serogroupC (MenC) capsular saccharide conjugated to tetanus toxoid carrierprotein (TT); a Neisseria meningitidis serogroup W135 (MenW) capsularsaccharide conjugated to tetanus toxoid carrier protein (TT); and aNeisseria meningitidis serogroup Y (MenY) capsular saccharide conjugatedto tetanus toxoid carrier protein (TT).

In one aspect, the invention relates to a method for eliciting an immuneresponse in a patient of any age. The method includes administering tothe human a composition including a first lipidated polypeptideincluding the amino acid sequence set forth in SEQ ID NO: 1; a secondlipidated polypeptide including the amino acid sequence set forth in SEQID NO: 2. In one embodiment, the composition further includespolysorbate-80. In one embodiment, the composition further includesaluminum. In one embodiment, the composition further includes histidine.In one embodiment, the composition further includes sodium chloride. Inone embodiment, the composition further includes polysorbate-80,aluminum, histidine, and sodium chloride. In yet another embodiment, thecomposition further includes a Neisseria meningitidis serogroup A (MenA)capsular saccharide conjugated to tetanus toxoid carrier protein (TT); aNeisseria meningitidis serogroup C (MenC) capsular saccharide conjugatedto tetanus toxoid carrier protein (TT); a Neisseria meningitidisserogroup W135 (MenW) capsular saccharide conjugated to tetanus toxoidcarrier protein (TT); and a Neisseria meningitidis serogroup Y (MenY)capsular saccharide conjugated to tetanus toxoid carrier protein (TT).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—Overlaid IEX-HPC Chromatograms for the MnB bivalent rLP2086composition in the Absence and Presence of the MenACWY-TT composition,described in Example 6.

FIG. 2—Overlay of RP-HPLC Chromatograms showing that the presence of theMenACWY-TT composition does not interfere with evaluation of the MnBbivalent rLP2086 composition purity, as described in Example 7.

FIG. 3—Overlay of RP-HPLC Chromatograms showing rLP2086 Protein Purityand Peak Ratio in the Combined MenABCWY composition, as described inExample 14.

FIG. 4A—Primary Amino Acid Sequence of MnB rLP2086 Subfamily A A05Protein and

FIG. 4B—Primary Structure of MnB rLP2086 Subfamily A A05 Protein

FIG. 5A—Primary Amino Acid Sequence of MnB rLP2086 Subfamily B B01Protein and

FIG. 5B—Primary Structure of MnB rLP2086 Subfamily B B01 Protein

FIG. 6A—Amino acid sequences for a factor H binding protein (fHBP) B16(SEQ ID NO: 26) from a N. meningitidis serogroup A strain (fHBP variantB16 (PMB3257, MenA);

FIG. 6B—a fHBP A10 (SEQ ID NO: 27) from a N. meningitidis serogroup Cstrain (fHBP variant A10 (PMB5208, MenC and PMB5523, MenW);

FIG. 6C—a fHBP A19 (SEQ ID NO: 28) from a N. meningitidis serogroup Wstrain (fHBP variant A19 (PMB5248, MenW);

FIG. 6D—a fHBP A10 (SEQ ID NO: 27) from a N. meningitidis serogroup Wstrain (fHBP variant A10 (PMB5523, MenW);

FIG. 6E—a fHBP B47 (SEQ ID NO: 29) from a N. meningitidis serogroup Ystrain (fHBP variant B47 (PMB5187, MenY);

FIG. 6F—a fHBP B49 (SEQ ID NO: 30) from a N. meningitidis serogroup Xstrain (fHBP variant B49 (PMB5540, MenX).

FIG. 7—Serum Bactericidal Activity, the Correlate of Protection forMeningococcal Disease. A titer of ≥1:4 in serum bactericidal assaysusing human complement (hSBA) is the established correlate of protectionfor meningococcal disease.

FIG. 8—MenA, C, W, Y, and X Test Strain Selection

FIG. 9—Schematic of the Relevant Groups of the Clinical Trial From Whicha Subset of Test Sera Were Randomly Selected

FIG. 10—Distribution of FHbp Surface Expression Levels (MFI) DeterminedFrom Flow Cytometric Experiments Using the FHbp Reactive mAb MN 994-11.The FHbp surface expression for each of the strains within a serogroupis noted with a black dot while the FHbp surface expression levels forthe selected test strains within each serogroup are noted with a coloredstar.

FIG. 11—hSBA Response Rate (Percentage of Subjects With hSBA Titers≥1:8) for MenA PMB3257 (B16). Response rates and 95% confidenceintervals for sera collected at preimmunization (month 0) and 1 monthafter doses 1, 2, and 3 for MenBFHbp are shown. The geometric meantiters (GMTs) obtained were 2, 3, 4, and 5, respectively. The responserates for subjects in the positive control group were 3% prior tovaccination and 97% one month after receiving MCV4. The GMTs for thepositive control group were 2 and 95, respectively.

FIG. 12—hSBA Response Rate (Percentage of Subjects With hSBA Titers≥1:8) for MenC PMB5208 (A10). Response rates and 95% confidenceintervals for sera collected at preimmunization (month 0) and 1 monthafter doses 1, 2, and 3 for MenBFHbp are shown. The GMTs obtained were4, 8, 12, and 29, respectively. The response rates for subjects in thepositive control group were 20% prior to vaccination and 90% one monthafter receiving MCV4. The GMTs for the positive control group were 3 and119, respectively.

FIG. 13—hSBA Response Rate (Percentage of Subjects With hSBA Titers≥1:8) for MenW PMB5248 (A19). Response rates and 95% confidenceintervals for sera collected at preimmunization (month 0) and 1 monthafter doses 1, 2, and 3 for MenBFHbp are shown. The GMTs obtained were4, 18, 47, and 77, respectively. The response rates for subjects in thepositive control group were 40% prior to vaccination and 97% one monthafter receiving MCV4. The GMTs for the positive control group were 5 and88, respectively.

FIG. 14—hSBA Response Rate (Percentage of Subjects With hSBA Titers≥1:8) for MenW PMB5523 (A10). Response rates and 95% confidenceintervals for sera collected at preimmunization (month 0) and 1 monthafter doses 1, 2, and 3 for MenBFHbp are shown. The GMTs obtained were7, 15, 21, and 42, respectively. The response rates for subjects in thepositive control group were 55% prior to vaccination and 97% one monthafter receiving MCV4. The GMTs for the positive control group were 8 and60, respectively.

FIG. 15—hSBA Response Rate (Percentage of Subjects With hSBA Titers≥1:8) for MenY PMB5187 (B47). Response rates and 95% confidenceintervals for sera collected at preimmunization (month 0) and 1 monthafter doses 1, 2, and 3 for MenBFHbp are shown. The GMTs obtained were3, 7, 31, and 58, respectively. The response rates for subjects in thepositive control group were 13% prior to vaccination and 97% one monthafter receiving MCV4. The GMTs for the positive control group were 3 and79, respectively.

FIG. 16—hSBA Response Rate (Percentage of Subjects With hSBA Titers≥1:8) for MenX PMB5540 (B49). Response rates and 95% confidenceintervals for sera collected at preimmunization (month 0) and 1 monthafter doses 1, 2, and 3 for MenBFHbp are shown. The GMTs obtained were2, 3, 7, and 20, respectively. The response rates for subjects in thepositive control group were 0% prior to vaccination and 0% one monthafter receiving MCV4 vaccine. The GMTs for the positive control groupwere 2 and 2, respectively.

SEQUENCE IDENTIFIERS

SEQ ID NO: 1 sets forth the amino acid sequence for a recombinant N.meningitidis, serogroup B, 2086 variant A05 polypeptide antigen.

SEQ ID NO: 2 sets forth the amino acid sequence for a recombinant N.meningitidis, serogroup B, 2086 variant B01 polypeptide antigen.

SEQ ID NO: 3 sets forth the amino acid residues at positions 1-4 of SEQID NO: 1 and SEQ ID NO: 2.

SEQ ID NO: 4 sets forth the amino acid sequence of the N-terminus of arecombinant Neisserial Subfamily A LP2086 polypeptide (rLP2086) (A05)polypeptide.

SEQ ID NO: 5 sets forth the amino acid sequence of the N-terminus ofNeisserial Subfamily A LP2086 M98250771 polypeptide (A05) polypeptide.

SEQ ID NO: 6 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant B153.

SEQ ID NO: 7 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant A04.

SEQ ID NO: 8 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant A05

SEQ ID NO: 9 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant A12.

SEQ ID NO: 10 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant A22.

SEQ ID NO: 11 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant B02.

SEQ ID NO: 12 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant B03.

SEQ ID NO: 13 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant B09.

SEQ ID NO: 14 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant B22.

SEQ ID NO: 15 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant B24.

SEQ ID NO: 16 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant B44.

SEQ ID NO: 17 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant B16.

SEQ ID NO: 18 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant A07.

SEQ ID NO: 19 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant A19.

SEQ ID NO: 20 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant A06.

SEQ ID NO: 21 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant A15.

SEQ ID NO: 22 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant A29.

SEQ ID NO: 23 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant B15.

SEQ ID NO: 24 sets forth the amino acid sequence of the N-terminus of arecombinant Neisserial Subfamily B LP2086 polypeptide (rLP2086) (B01)polypeptide.

SEQ ID NO: 25 sets forth the amino acid sequence of the N-terminus ofNeisserial Subfamily B LP2086 CDC-1573 polypeptide (B01) polypeptide.

SEQ ID NO: 26 sets forth the amino acid sequence for N. meningitidisserogroup A strain expressing factor H binding protein (fHBP) B16.

SEQ ID NO: 27 sets forth the amino acid sequence for a N. meningitidisserogroup C strain expressing fHBP A10. SEQ ID NO: 27 also sets forththe amino acid sequence for a N. meningitidis serogroup W strainexpressing fHBP A10.

SEQ ID NO: 28 sets forth the amino acid sequence for a N. meningitidisserogroup W strain expressing fHBP A19.

SEQ ID NO: 29 sets forth the amino acid sequence for a N. meningitidisserogroup Y strain expressing fHBP B47.

SEQ ID NO: 30 sets forth the amino acid sequence for a N. meningitidisserogroup X strain expressing fHBP B49.

SEQ ID NO: 31 sets forth the amino acid sequence for a non-lipidated N.meningitidis, serogroup B, 2086 variant B16.

SEQ ID NO: 32 sets forth the amino acid sequence for a non-lipidated N.meningitidis, serogroup B, 2086 variant A07.

SEQ ID NO: 33 sets forth the amino acid sequence for a non-lipidated N.meningitidis, serogroup B, 2086 variant A19.

SEQ ID NO: 34 sets forth the amino acid sequence for a non-lipidated N.meningitidis, serogroup B, 2086 variant A06.

SEQ ID NO: 35 sets forth the amino acid sequence for a non-lipidated N.meningitidis, serogroup B, 2086 variant A15.

SEQ ID NO: 36 sets forth the amino acid sequence for a non-lipidated N.meningitidis, serogroup B, 2086 variant A29.

SEQ ID NO: 37 sets forth the amino acid sequence for a non-lipidated N.meningitidis, serogroup B, 2086 variant B15.

SEQ ID NO: 38 sets forth the amino acid sequence for a non-lipidated N.meningitidis serogroup A strain expressing factor H binding protein(fHBP) B16.

SEQ ID NO: 39 sets forth the amino acid sequence for a non-lipidated N.meningitidis serogroup C strain expressing fHBP A10. SEQ ID NO: 39 alsosets forth the amino acid sequence for a non-lipidated N. meningitidisserogroup W strain expressing fHBP A10.

SEQ ID NO: 40 sets forth the amino acid sequence for a non-lipidated N.meningitidis serogroup W strain expressing fHBP A19.

SEQ ID NO: 41 sets forth the amino acid sequence for a non-lipidated N.meningitidis serogroup Y strain expressing fHBP B47.

SEQ ID NO: 42 sets forth the amino acid sequence for a non-lipidated N.meningitidis serogroup X strain expressing fHBP B49.

SEQ ID NO: 43 sets forth the amino acid sequence for a non-lipidated N.meningitidis, serogroup B, 2086 variant B44.

SEQ ID NO: 44 sets forth the amino acid sequence for a non-lipidated N.meningitidis, serogroup B, 2086 variant B09.

SEQ ID NO: 45 sets forth the amino acid sequence for a N. meningitidis,serogroup B, 2086 variant B09.

SEQ ID NO: 46 sets forth the amino acid sequence for a non-lipidated N.meningitidis, serogroup B, 2086 variant A05.

SEQ ID NO: 47 sets forth the amino acid sequence for a non-lipidated N.meningitidis, serogroup B, 2086 variant B01.

SEQ ID NO: 48 sets forth the amino acid sequence for a N. meningitidis,serogroup B, 2086 variant B01, which includes an N-terminal Cys at aminoacid position 1.

SEQ ID NO: 49 sets forth the amino acid sequence for a N. meningitidis,serogroup B, 2086 variant B15, which includes an N-terminal Cys at aminoacid position 1.

SEQ ID NO: 50 sets forth the amino acid sequence for a N. meningitidis,serogroup B, 2086 variant B16, which includes an N-terminal Cys at aminoacid position 1.

SEQ ID NO: 51 sets forth the amino acid sequence for a N. meningitidis,serogroup B, 2086 variant B22.

SEQ ID NO: 52 sets forth the amino acid sequence for a N. meningitidis,serogroup B, 2086 variant A22.

SEQ ID NO: 53 sets forth the amino acid sequence for a non-lipidated N.meningitidis, serogroup B, 2086 variant A12.

SEQ ID NO: 54 sets forth the amino acid sequence for a non-lipidated N.meningitidis, serogroup B, 2086 variant A22.

SEQ ID NO: 55 sets forth the amino acid sequence for a N. meningitidisserogroup B, 2086 variant A62, which includes an N-terminal Cys at aminoacid position 1.

SEQ ID NO: 56 sets forth the amino acid sequence for a non-lipidated N.meningitidis, serogroup B, 2086 variant A62.

SEQ ID NO: 57 sets forth the amino acid sequence for a N. meningitidis,serogroup B, 2086 variant A29, which includes an N-terminal Cys at aminoacid position 1.

SEQ ID NO: 58 sets forth the amino acid sequence for a non-lipidated N.meningitidis, serogroup B, 2086 variant B22.

SEQ ID NO: 59 sets forth the amino acid sequence for a N. meningitidis,serogroup B, 2086 variant A05.

SEQ ID NO: 60 sets forth the amino acid sequence for a non-lipidated N.meningitidis, serogroup B, 2086 variant A05.

SEQ ID NO: 61 sets forth the amino acid sequence for a N. meningitidis,serogroup B, 2086 variant B24.

SEQ ID NO: 62 sets forth the amino acid sequence for a N. meningitidis,serogroup B, 2086 variant B24.

DETAILED DESCRIPTION OF THE INVENTION

The inventors surprisingly discovered a composition including at leastone factor H binding protein (fHBP) and at least one N. meningitidiscapsular saccharide conjugate. The composition is surprisingly stableand elicited an immune response against strains that express fHBPvariants that are homologous to the fHBP variant in the multi-componentcomposition and an immune response against strains that express fHBPvariants that are heterologous to the fHBP variant in themulti-component composition. The inventors further surprisinglydiscovered that an fHBP polypeptide effectively elicited an immuneresponse in children, such as, for example, humans aged 12 months andabove. Moreover, the inventors surprisingly discovered that an fHBPpolypeptide effectively elicited an immune response against a N.meningitidis serogroup X strain.

The inventors surprisingly discovered a composition that includes (a) afirst lipidated polypeptide including the amino acid sequence set forthin SEQ ID NO: 1; (b) a second lipidated polypeptide including the aminoacid sequence set forth in SEQ ID NO: 2; (c) a Neisseria meningitidisserogroup A (MenA) capsular saccharide conjugated to an adipic aciddihydrazide (ADH) linker by 1-cyano-4-dimethylamino pyridiniumtetrafluoroborate chemistry, wherein the linker is conjugated to tetanustoxoid carrier protein (TT) by carbodiimide chemistry (MenA_(AH)-TTconjugate); (d) a Neisseria meningitidis serogroup C (MenC) capsularsaccharide conjugated to an ADH linker by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry, wherein the linker is conjugatedto tetanus toxoid carrier protein (TT) by carbodiimide chemistry(MenC_(AH)-TT conjugate); (e) a Neisseria meningitidis serogroup W135(MenW) capsular saccharide directly conjugated to tetanus toxoid carrierprotein (TT) by 1-cyano-4-dimethylamino pyridinium tetrafluoroboratechemistry, in the absence of a linker (MenW-TT conjugate); (f) aNeisseria meningitidis serogroup Y (MenY) capsular saccharide directlyconjugated to tetanus toxoid carrier protein (TT) by1-cyano-4-dimethylamino pyridinium tetrafluoroborate chemistry, in theabsence of a linker (MenY-TT conjugate). The composition includes alyophilized MenACWY-TT composition that surprisingly is readilyreconstituted with a liquid MnB bivalent rLP2086 composition, whereinthe composition is in a single vial. The inventors discovered that thelyophilized MenACWY-TT composition and the liquid MnB bivalent rLP2086composition were compatible and stable, following reconstitution, for atleast 24 hours at room temperature.

Moreover, the inventors further discovered that the MnB bivalent rLP2086composition elicited bactericidal antibodies not only against N.meningitidis serogroup B, but also N. meningitidis serogroups other thanB. For example, the MnB bivalent rLP2086 composition elicitedbactericidal antibodies against at least N. meningitidis serogroups A,C, W, Y, and X. The surprising discovery that the MnB bivalent rLP2086composition elicited bactericidal antibodies against N. meningitidisserogroup X indicates that the MnB bivalent rLP2086 composition elicitsa broadly cross-reactive bactericidal immune response in humans againstat least two diverse Neisseria meningitidis serogroups.

Furthermore, the inventors surprisingly discovered an immune response asmeasured by serum bactericidal assay using human complement (hSBA)performed with 4 primary Neisseria meningitidis serogroup B (MnB) teststrains, 2 expressing an LP2086 subfamily A protein and 2 expressing anLP2086 subfamily B protein, measured 1 month after the third vaccinationwith bivalent rLP2086, in healthy subjects aged ≥24 months to <4 yearsat study entry. The inventors also surprisingly discovered an immuneresponse as measured by hSBA performed with 4 primary MnB test strains,2 expressing an LP2086 subfamily A protein and 2 expressing an LP2086subfamily B protein, measured 1 month after the third vaccination withbivalent rLP2086, in healthy subjects aged ≥4 years to <10 years atstudy entry. The inventors further surprisingly discovered an immuneresponse as measured by hSBA performed with 4 primary MnB test strains,2 expressing an LP2086 subfamily A protein and 2 expressing an LP2086subfamily B protein, measured 1 month after the third vaccination withbivalent rLP2086, in healthy subjects aged ≥24 months to <10 years atstudy entry (ie, in the combined age stratum). The inventors alsosurprisingly discovered an immune response as measured by hSBA performedwith 4 primary MnB test strains, 2 expressing an LP2086 subfamily Aprotein and 2 expressing an LP2086 subfamily B protein, measured 1 monthafter the second vaccination and 6 months after the third vaccinationwith bivalent rLP2086, in healthy subjects aged ≥24 months to <4 yearsat study entry, in healthy subjects aged ≥4 years to <10 years at studyentry, and in the combined age stratum. The immune response was furtherdescribed through additional endpoints, as measured by hSBA performedwith 4 primary MnB test strains, 2 expressing an LP2086 subfamily Aprotein and 2 expressing an LP2086 subfamily B protein, at specifiedtime points, in healthy subjects aged ≥24 months to <4 years at studyentry, in healthy subjects aged ≥4 years to <10 years at study entry,and in the combined age stratum.

In addition, the inventors surprisingly discovered an immune response asmeasured by serum bactericidal assay using human complement (hSBA)performed with 4 primary Neisseria meningitidis serogroup B (MnB)strains, 2 expressing an LP2086 subfamily A protein and 2 expressing anLP2086 subfamily B protein, measured 1 month after the third vaccinationwith bivalent rLP2086, in healthy toddlers aged 12 to <18 months atstudy entry. The inventors also surprisingly discovered an immuneresponse as measured by hSBA performed with 4 primary MnB strains, 2expressing an LP2086 subfamily A protein and 2 expressing an LP2086subfamily B protein, measured 1 month after the third vaccination withbivalent rLP2086, in healthy toddlers aged 18 to <24 months at studyentry. The inventors further surprisingly discovered an immune responseas measured by hSBA performed with 4 primary MnB test strains, 2expressing an LP2086 subfamily A protein and 2 expressing an LP2086subfamily B protein, measured 1 month after the third vaccination withbivalent rLP2086, in healthy toddlers aged 12 to <24 months at studyentry (ie, both age strata combined). The inventors also surprisinglydiscovered an immune response as measured by hSBA performed with 4primary MnB test strains, 2 expressing an LP2086 subfamily A protein and2 expressing an LP2086 subfamily B protein, measured 1 month after thesecond vaccination and at least 6 months after the third vaccination inhealthy toddlers aged 12 to <18 months and 18 to <24 months at studyentry, and in both age strata combined. For example, the hSBA may bemeasured at any time, including 12, 24, 36, and 48 months after thethird vaccination in healthy toddlers aged 12 to <18 months and 18 to<24 months at study entry, and in both age strata combined. The immuneresponse was further described through additional endpoints, as measuredby hSBA performed with 4 primary MnB test strains, 2 expressing anLP2086 subfamily A protein and 2 expressing an LP2086 subfamily Bprotein, measured 1 month after the second vaccination and at least 1month after the third vaccination with bivalent rLP2086 in healthytoddlers aged 12 to <18 months and 18 to <24 months at study entry, andin both age strata combined. For example, the hSBA may be measured atany time, including 6, 12, 24, 36, and 48 months after the thirdvaccination with bivalent rLP2086 in healthy toddlers aged 12 to <18months and 18 to <24 months at study entry, and in both age stratacombined. The immune response was also described through additionalendpoints, as measured by hSBA to secondary MnB test strains expressingLP2086 subfamily A and B proteins, at 1 month after the secondvaccination and at least 1 month after the third vaccination in healthytoddlers aged 12 to <18 months and 18 to <24 months at study entry, andin both age strata combined. For example, the hSBA may be measured atany time, including 6, 12, 24, 36, and 48 months after the thirdvaccination in healthy toddlers aged 12 to <18 months and 18 to <24months at study entry, and in both age strata combined.

Accordingly, in one aspect, the invention relates to a method foreliciting an immune response in a patient of any age. In someembodiments, the human is aged at least 4 weeks, 5 weeks, 6 weeks, 7weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks old. Forexample, in a preferred embodiment, the human is aged at least 6 weeks.As is known in the art, a Meningococcal Group A, C, W-135, and YConjugate Vaccine, such as NIMENRIX 0, is suitable for infants as earlyas six weeks of age, and can be administered to any human aged six weeksand above. In some embodiments, the human is aged at least 6 months, 7months, 8 months, 9 months, 10 months, 11 months, or 12 months. Forexample, in a preferred embodiment, the human is aged at least 12months. In one embodiment, the human is aged between 12 and 18 months.In another aspect, the invention relates to a method for eliciting animmune response in a patient aged at least 18 months. In one embodiment,the human is aged between 18 and 24 months. In yet another aspect, theinvention relates to a method for eliciting an immune response in apatient aged at least 24 months. In one embodiment, the human is agedbetween 24 months and 10 years. In another aspect, the invention relatesto a method for eliciting an immune response in a patient aged 10 yearsand above. In a further aspect, the invention relates to a method foreliciting an immune response in a patient aged between 10 years and 25years. The method includes administering to the human a compositionincluding a first lipidated polypeptide including the amino acidsequence set forth in SEQ ID NO: 1; a second lipidated polypeptideincluding the amino acid sequence set forth in SEQ ID NO: 2. In oneembodiment, the composition further includes polysorbate-80. In oneembodiment, the composition further includes aluminum. In oneembodiment, the composition further includes histidine. In oneembodiment, the composition further includes sodium chloride. In oneembodiment, the composition further includes polysorbate-80, aluminum,histidine, and sodium chloride. In yet another embodiment, thecomposition further includes a Neisseria meningitidis serogroup A (MenA)capsular saccharide conjugated to tetanus toxoid carrier protein (TT); aNeisseria meningitidis serogroup C (MenC) capsular saccharide conjugatedto tetanus toxoid carrier protein (TT); a Neisseria meningitidisserogroup W135 (MenW) capsular saccharide conjugated to tetanus toxoidcarrier protein (TT); and a Neisseria meningitidis serogroup Y (MenY)capsular saccharide conjugated to tetanus toxoid carrier protein (TT).As is known in the art, a Meningococcal Group A, C, W-135, and YConjugate Vaccine, such as NIMENRIX®, is suitable for infants as earlyas six weeks of age, and can be administered to any human aged six weeksand above.

Further descriptions of exemplary compositions are described below.

Composition and Vaccine

The inventors further discovered that a composition including fHBPelicits an effective immune response in humans aged at least 12 months.The composition also elicits an immune response against a N.meningitidis serogroup X strain. In addition, the inventors surprisinglydiscovered a composition including at least one factor H bindingpolypeptide (fHBP) and at least one N. meningitidis capsular saccharideconjugate. The composition is surprisingly stable and elicited an immuneresponse against strains that express fHBP variants that are homologousto the fHBP variant in the multi-component composition and an immuneresponse against strains that express fHBP variants that areheterologous to the fHBP variant in the multi-component composition. Inone embodiment, the composition includes any fHBP, such as, for example,any one of the following polypeptides: B24, B16, B44, A22, B03, B09,A12, A19, A05, A07, A06, A15, A29, B01, A62, B15, and any combinationthereof. Preferably, the composition includes a combination of A05 andB01 polypeptides. In another preferred embodiment, the compositionincludes a combination of B24 and A05 polypeptides. In anotherembodiment, the composition includes a combination of A05, A12, B09, andB44 polypeptides. In one embodiment, the composition includes alipidated fHBP. In one embodiment, the composition does not include anon-lipidated fHBP.

In another embodiment, the composition includes a non-lipidated fHBP,such as any one of the non-lipidated fHBP described in InternationalPatent Publication No. WO2012/032489, US Patent Publication No.US20120093852, International Patent Publication No. WO2013/132452, andUS Patent Publication No. US20160030543, which are each incorporatedherein by reference in their entirety. In one embodiment, thecomposition includes at least one non-lipidated fHBP and at least onelipidated fHBP.

In some embodiments, the composition includes a polypeptide having atleast 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 99.9% identity to the amino acid sequence set forth inany one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 7, SEQID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17,SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO:22, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ IDNO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38,SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO:43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ IDNO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57,SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, and SEQ IDNO: 62.

The inventors further surprisingly discovered that a liquid MnB bivalentrLP2086 composition can readily reconstitute a lyophilized MenACWY-TTcomposition and that the combined composition is compatible and stable.

In one aspect, the invention relates to a composition against Neisseriameningitidis. The composition includes (a) a first lipidated polypeptideincluding the amino acid sequence set forth in SEQ ID NO: 1; (b) asecond lipidated polypeptide including the amino acid sequence set forthin SEQ ID NO: 2; (c) a Neisseria meningitidis serogroup A (MenA)capsular saccharide conjugated to an adipic acid dihydrazide (ADH)linker by 1-cyano-4-dimethylamino pyridinium tetrafluoroboratechemistry, wherein the linker is conjugated to tetanus toxoid carrierprotein (TT) by carbodiimide chemistry (MenA_(AH)-TT conjugate); (d) aNeisseria meningitidis serogroup C (MenC) capsular saccharide conjugatedto an ADH linker by 1-cyano-4-dimethylamino pyridinium tetrafluoroboratechemistry, wherein the linker is conjugated to tetanus toxoid carrierprotein (TT) by carbodiimide chemistry (MenC_(AH)-TT conjugate); (e) aNeisseria meningitidis serogroup W135 (MenW) capsular saccharidedirectly conjugated to tetanus toxoid carrier protein (TT) by1-cyano-4-dimethylamino pyridinium tetrafluoroborate chemistry, in theabsence of a linker (MenW-TT conjugate); (f) a Neisseria meningitidisserogroup Y (MenY) capsular saccharide directly conjugated to tetanustoxoid carrier protein (TT) by 1-cyano-4-dimethylamino pyridiniumtetrafluoroborate chemistry, in the absence of a linker (MenY-TTconjugate).

In another aspect, the invention relates to a composition that includesa combination of a MnB bivalent rLP2086 composition and a MenACWY-TTcomposition. The MnB bivalent rLP2086 composition refers to acomposition that includes a single N. meningitidis polypeptide componentthat induces an effective broadly protective immune response againstmultiple strains of N. meningitidis serogroup B. Specifically, in oneembodiment, the MnB bivalent rLP2086 composition includes a MnB rLP2086subfamily A protein (SEQ ID NO: 1) and MnB rLP2086 subfamily B protein(SEQ ID NO: 2). In one embodiment, the composition does not include afusion protein. In one embodiment, the composition does not include achimeric protein. In one embodiment, the composition does not include ahybrid protein. In one embodiment, the composition does not furtherinclude a peptide fragment. In another embodiment, the composition doesnot further include a Neisserial polypeptide that is not fHBP. Forexample, in one embodiment, the composition does not include a PorAprotein. In another embodiment, the composition does not include a NadAprotein. In another embodiment, the composition does not further includea Neisserial heparin binding antigen (NHBA). In another embodiment, thecomposition does not further include a Neisserial outer membrane vesicle(OMV). In a preferred embodiment, the composition does not furtherinclude antigens, other than the first polypeptide and the secondpolypeptide. In a preferred embodiment, the MnB bivalent rLP2086composition further includes polysorbate-80. In one embodiment, the MnBbivalent rLP2086 composition further includes histidine buffer. In oneembodiment, the MnB bivalent rLP2086 composition further includes sodiumchloride. In one embodiment, the MnB bivalent rLP2086 compositionfurther includes aluminum phosphate. In one embodiment, the MnB bivalentrLP2086 composition further includes polysorbate-80, histidine buffer,sodium chloride, and aluminum phosphate. Preferably, the MnB bivalentrLP2086 composition is a liquid formulation, wherein the polypeptidesare formulated as 120 mcg/mL/subfamily in 10 mM histidine buffer, pH6.0, 150 mM sodium chloride (NaCl) with 0.5 mg/mL aluminum phosphate(AlPO₄), and further includes 0.018 mg polysorbate-80 in a 0.5 mL dose.

The MenACWY-TT composition refers to a composition that includespurified capsular polysaccharides of Neisseria meningitidis Serogroup A,C, W-135 and Y, each independently conjugated to TT at ratios (TT topolysaccharide) of ˜3, ˜3, ˜1.5 and ˜1.3, respectively. Specifically,the composition includes (c) a Neisseria meningitidis serogroup A (MenA)capsular saccharide conjugated to an adipic acid dihydrazide (ADH)linker by 1-cyano-4-dimethylamino pyridinium tetrafluoroboratechemistry, wherein the linker is conjugated to tetanus toxoid carrierprotein (TT) by carbodiimide chemistry (MenA_(AH)-TT conjugate); (d) aNeisseria meningitidis serogroup C (MenC) capsular saccharide conjugatedto an ADH linker by 1-cyano-4-dimethylamino pyridinium tetrafluoroboratechemistry, wherein the linker is conjugated to tetanus toxoid carrierprotein (TT) by carbodiimide chemistry (MenC_(AH)-TT conjugate); (e) aNeisseria meningitidis serogroup W135 (MenW) capsular saccharidedirectly conjugated to tetanus toxoid carrier protein (TT) by1-cyano-4-dimethylamino pyridinium tetrafluoroborate chemistry, in theabsence of a linker (MenW-TT conjugate); (f) a Neisseria meningitidisserogroup Y (MenY) capsular saccharide directly conjugated to tetanustoxoid carrier protein (TT) by 1-cyano-4-dimethylamino pyridiniumtetrafluoroborate chemistry, in the absence of a linker (MenY-TTconjugate). Preferably, the MenACWY-TT composition is presented as alyophilized powder.

MenA_(AH)-TT, MenC_(AH)-TT, MenW-TT, and MenY-TT conjugates are preparedthrough the following steps: manufacture of the polysaccharide drugsubstance intermediate, manufacture of the TT drug substanceintermediate, microfluidization of the polysaccharide, derivatization ofthe polysaccharide (for the MenAAH-TT and MenCAH-TT processes only),additional purification of the TT, and conjugation of the individualpolysaccharides to TT.

Regarding the MenA_(AH)-TT conjugate, the MenA polysaccharide is firstmicrofluidized to reduce molecular size and viscosity, then activatedvia cyanylation with 1-cyano-4-dimethylamino-pyridiniumtetrafluoroborate (CDAP). Activated MenA is derivatized with adipic aciddihydrazide (ADH) to form the MenA_(AH). MenAAH and Tetanus Toxoid (TT)are coupled through carbodiimide-mediated condensation(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) couplingtechnology) to form MenAAH-Tetanus Toxoid Conjugate (MenA_(AH)-TT).

Regarding the MenC_(AH)-TT conjugate, the MenC polysaccharide is firstmicrofluidized to reduce molecular size and viscosity, then activatedvia cyanylation with CDAP. Activated MenC is derivatized with adipicacid dihydrazide (ADH) to form the MenC_(AH). MenC_(AH) and TT arecoupled through carbodiimide-mediated condensation EDAC couplingtechnology) to form MenC_(AH)-Tetanus Toxoid (MenC_(AH)-TT).

Regarding the MenW-TT conjugate, MenW polysaccharide is firstmicrofluidized to reduce molecular size and viscosity, then activatedvia cyanylation with CDAP. Activated MenW is directly coupled to TT toform MenW-Tetanus Toxoid (MenW-TT).

Regarding the MenY-TT conjugate, MenY polysaccharide is firstmicrofluidized to reduce molecular size and viscosity, then activatedvia cyanylation with CDAP. Activated MenY is directly coupled to TT toform MenY-Tetanus Toxoid (MenY-TT).

In another aspect, the inventors surprisingly discovered thatpolypeptide antigens derived from at most two N. meningitidis serogroupB strains induces an effective broadly protective immune responseagainst multiple strains of N. meningitidis serogroup B. Accordingly, inone embodiment, the composition does not further include a polypeptidethat is not derived from N. meningitidis serogroup B fHBP subfamily AM98250771 strain and/or N. meningitidis serogroup B fHBP subfamily BCDC1573 strain.

In one embodiment, the composition does not further include apolypeptide having less than 100% sequence identity to SEQ ID NO: 1. Inanother embodiment, the composition does not further include apolypeptide having less than 100% sequence identity to SEQ ID NO: 2. Forexample, the composition does not further include a polypeptide havingless than 100% sequence identity to the full length of SEQ ID NO: 1and/or SEQ ID NO: 2.

In one embodiment, the composition further includes polysorbate-80,aluminum, histidine, and sodium chloride. In one embodiment, thecomposition includes about 60 μg of a first lipidated polypeptideincluding the amino acid sequence set forth in SEQ ID NO: 1, about 60 μgof a second lipidated polypeptide including the amino acid sequence setforth in SEQ ID NO: 2, 2.8 molar ratio of polysorbate-80 to eachpolypeptide, 0.5 mg aluminum/ml as aluminum phosphate, 10 mM histidine,and 150 mM sodium chloride, wherein the composition preferably has atotal volume of about 0.5 ml.

In another aspect, the composition includes about 120 μg/ml of a firstlipidated polypeptide including the amino acid sequence set forth in SEQID NO: 1, about 120 μg/ml of a second lipidated polypeptide includingthe amino acid sequence set forth in SEQ ID NO: 2, 2.8 molar ratio ofpolysorbate-80 to each polypeptide, 0.5 mg aluminum/ml as aluminumphosphate, 10 mM histidine, and 150 mM sodium chloride.

In a further aspect, the composition includes a) 60 μg of a firstlipidated polypeptide including the amino acid sequence set forth in SEQID NO: 1; b) 60 μg of a second lipidated polypeptide including the aminoacid sequence set forth in SEQ ID NO: 2; c) 18 μg polysorbate-80; d) 250μg aluminum; e) 780 μg histidine, and; f) 4380 μg sodium chloride.

In an exemplary embodiment, the composition includes about 60 μg of afirst lipidated polypeptide consisting of the amino acid sequence setforth in SEQ ID NO: 1, about 60 μg of a second lipidated polypeptideconsisting of the amino acid sequence set forth in SEQ ID NO: 2, 2.8molar ratio of polysorbate-80 to first lipidated polypeptide and tosecond lipidated polypeptide, 0.5 mg/ml aluminum phosphate, 10 mMhistidine, and 150 mM sodium chloride, wherein the composition has atotal volume of about 0.5 ml. In the exemplary embodiment, thecomposition is a sterile isotonic buffered liquid suspension. In theexemplary embodiment, the composition has a pH 6.0. In the exemplaryembodiment, the first polypeptide and the second polypeptide areadsorbed to aluminum.

In one embodiment, the composition includes a MenA_(AH)-TT conjugatehaving a mean TT/polysaccharide ratio 3; a MenC_(AH)-TT conjugate havinga mean TT/polysaccharide ratio 3; a MenW-TT conjugate having a meanTT/polysaccharide ratio 1.5; and a MenY-TT conjugate having a meanTT/polysaccharide ratio 1.3. In a preferred embodiment, the compositionincludes a MenA_(AH)-TT conjugate having 5 mcg MenA polysaccharide and˜15 mcg TT; a MenC_(AH)-TT conjugate having 5 mcg MenC polysaccharideand ˜15 mcg TT; a MenW-TT conjugate having 5 mcg MenW polysaccharide and˜7.5 mcg TT; and a MenY-TT conjugate having 5 mcg MenY polysaccharideand ˜6.5 mcg TT. The composition may further include Tris-HCl, sucrose,and sodium chloride.

In another embodiment, the composition includes a MenA_(AH)-TTconjugate; MenC_(AH)-TT conjugate; MenW-TT conjugate; and MenY-TTconjugate, which includes MenA polysaccharide; MenC polysaccharide; MenWpolysaccharide; and MenY polysaccharide and TT carrier protein. Thecomposition may further include sucrose and Trometanol. For example, inone embodiment, the composition includes 10 μg/mL MenA polysaccharide;10 μg/mL MenC polysaccharide; 10 μg/mL MenW polysaccharide; and 10 μg/mLMenY polysaccharide; 88 μg/mL TT carrier protein; 164 mM sucrose; and1.6 mM Trometanol.

In one embodiment, the composition has a total volume of about 0.5 ml.In one embodiment, a first dose of the composition has a total volume ofabout 0.5 ml. A “first dose” refers to the dose of the composition thatis administered on Day 0. A “second dose” or “third dose” refers to thedose of the composition that is administered subsequently to the firstdose, which may or may not be the same amount as the first dose.

In one aspect, the invention relates to a liquid immunogenic compositionresulting from the lyophilized MenACWY-TT composition having beenreconstituted with the liquid MnB bivalent rLP2086 composition.Reconstitution refers to restoring a dry lyophilized composition to aliquid form by the addition of a liquid diluent. In one preferredembodiment, the liquid MnB bivalent rLP2086 composition is notconcomitantly administered, is not coadministered with, and is notsimultaneously administered with the lyophilized MenACWY-TT composition,wherein the lyophilized MenACWY-TT composition has been reconstitutedwith a liquid composition that is not the liquid MnB bivalent rLP2086composition. For example, in one preferred embombodiment, thelyophilized MenACWY-TT composition is not reconstituted with an aqueousdiluent consisting of sodium chloride and water and is not subsequentlyconcomitantly administered, is not coadministered with, and is notsimultaneously administered with with the liquid MnB bivalent rLP2086composition.

Rather, in a preferred embodiment, the lyophilized MenACWY-TTcomposition is administered with the MnB bivalent rLP2086 composition inone, i.e., a single, administration to the human. The resulting singleadministration (e.g., the MenABCWY composition) may result from the MnBbivalent rLP2086 composition, from a first container, being mixed withthe lyophilized MenACWY-TT composition, from a second container.Alternatively, single administration of the MenABCWY composition mayresult from one (single) container that includes the MnB bivalentrLP2086 composition and the lyophilized MenACWY-TT composition. Deliverydevices for vaccine or immunogenic compositions are known in the art. Inone embodiment, the MenABCWY composition is administered concomitantlywith any one of ibuprofen, paracetamol, and amoxicillin.

The composition is immunogenic after administration of a first dose to ahuman. In one embodiment, the first dose is about 0.5 ml in totalvolume.

The composition induces a bactericidal titer of serum immunoglobulinthat is at least greater than 1-fold higher, preferably at least 2-foldhigher, in the human after receiving the first dose than a bactericidaltiter of serum immunoglobulin in the human prior to receiving the firstdose, when measured under identical conditions in a serum bactericidalassay using human complement (hSBA).

The bactericidal titer or bactericidal immune response is against N.meningitidis serogroup B. In a preferred embodiment, the bactericidaltiter or bactericidal immune response is against a N. meningitidisserogroup B fHBP subfamily A strain and against a N. meningitidisserogroup B fHBP subfamily B strain. Most preferably, the bactericidaltiter or bactericidal immune response is at least against N.meningitidis serogroup B, fHBP subfamily B, B01 strain.

In one embodiment, the composition induces a bactericidal titer of serumimmunoglobulin that is at least greater than 1-fold, such as, forexample, at least 1.01-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold,5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold,13-fold, 14-fold, 15-fold, or 16-fold higher in the human afterreceiving a dose of the composition than a bactericidal titer of serumimmunoglobulin in the human prior to receiving said dose, when measuredunder identical conditions in a serum bactericidal assay using humancomplement.

In one embodiment, the composition is an immunogenic composition. In oneembodiment, the composition is an immunogenic composition for a human.In another embodiment, the composition is a vaccine. A “vaccine” refersto a composition that includes an antigen, which contains at least oneepitope that induces an immune response that is specific for thatantigen. The vaccine may be administered directly into the subject bysubcutaneous, oral, oronasal, or intranasal routes of administration.Preferably, the vaccine is administered intramuscularly. In oneembodiment, the composition is a human vaccine. In one embodiment, thecomposition is an immunogenic composition against N. meningitidis.

In one embodiment, the composition is a liquid composition. In apreferred embodiment, the composition is a liquid suspensioncomposition. In another preferred embodiment, the composition is notlyophilized.

First Polypeptide; MNB RLP2086 Subfamily A (A05) Protein

In one embodiment, the composition includes a first polypeptide havingthe amino acid sequence set forth in SEQ ID NO: 1. The polypeptide is amodified factor H binding protein (fHBP) from N. meningitidis strainM98250771. A description of fHBP is disclosed in WO2012032489 and USpatent publication US 2012/0093852, which are each incorporated byreference in their entirety. The polypeptide is N-terminally lipidatedwith three predominant fatty acids C16:0, C16:1, and C18:1 covalentlylinked at three positions of the polypeptide. The first polypeptideincludes a total of 258 amino acids.

The representative primary structure of the MnB rLP2086 A05 protein ispresented in FIG. 4. The primary structure of the protein is illustratedin FIG. 4 using a single letter notation for all amino acids except forthe N-terminal cysteine and glyceryl moieties (illustrated using fullchemical formula). This structure includes the primary structure of theprotein sequence in which the N-terminal cysteine residue is lipidated.The amino group of the N-terminal cysteine residue at the proteinN-terminus is attached to a fatty acid (R1) forming an amide linkage andthe cysteinyl sulfhydryl group is attached to a glycerol moietycontaining two ester-bound fatty acids (R2). The structure of R1 isdeduced to be hexadecanoic acid (C16:0) and the structures of R2 varydepending on the MnB rLP2086 isoforms.

The first polypeptide includes two modifications introduced in theN-terminal region of the polypeptide, as compared to the correspondingwild-type sequence from N. meningitidis strain M98250771. A glycine inthe second position is added as a consequence of introducing a cloningsite. A second modification includes the deletion of four amino acids.Accordingly, in one embodiment, the first polypeptide includes a C-G-S-Ssequence (SEQ ID NO: 3) at the N-terminus. See SEQ ID NO: 1, first fouramino acid residues.

The N-terminal differences between the first polypeptide sequence andthe wild-type Neisserial sequence is shown below. Accordingly, in oneembodiment, the first polypeptide includes at least the first 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, or more amino acid residues of the aminoacid sequence set forth in SEQ ID NO: 1. Preferably, the firstpolypeptide includes at least the first 4, more preferably at least thefirst 6, and most preferably, at least the first 8 amino acid residuesof SEQ ID NO: 1.

Comparison of Predicted N-Terminal Sequencesof Recombinant and Neisserial Subfamily A LP2086 PolypeptiderLP2086 M98250771 CGSS

GGGGVAAD (SEQ ID NO: 4) Neisserial LP2086 M98250771 C-SSGS-GSGGGGVAAD(SEQ ID NO: 5) >A05 (SEQ ID NO: 1)CGSSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAEKTFKVGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPSGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELASAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIREKVH EIGIAGKQ

In one embodiment, the first polypeptide includes the amino acidsequence set forth in SEQ ID NO: 1. In one embodiment, the firstpolypeptide has a total of 258 amino acids. In one embodiment, the firstpolypeptide does not include an amino acid sequence having less than100% sequence identity to SEQ ID NO: 1. In another embodiment, the firstpolypeptide consists of the amino acid sequence set forth in SEQ IDNO: 1. In another embodiment, the first polypeptide includes the aminoacid sequence KDN. See for example, amino acid residues 73-75 of SEQ IDNO: 1. In another embodiment, the first polypeptide includes the aminoacid sequence set forth in SEQ ID NO: 3 at the N-terminus of thepolypeptide. In another embodiment, the first polypeptide includes theamino acid sequence set forth in SEQ ID NO: 4 at the N-terminus of thepolypeptide.

In a preferred embodiment, the first polypeptide is readily expressed ina recombinant host cell using standard techniques known in the art. Inanother preferred embodiment, the first polypeptide includes abactericidal epitope on the N- and/or C-domain of SEQ ID NO: 1. In oneembodiment, the first polypeptide includes at least the first 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, or 100 amino acid residues of the amino acid sequence set forthin SEQ ID NO: 1. Preferably, the first polypeptide includes at least thefirst 2, more preferably at least the first 4, and most preferably, atleast the first 8 amino acid residues of SEQ ID NO: 1.

In another embodiment, the first polypeptide includes at least the last4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, or 100 amino acid residues of the amino acidsequence set forth in SEQ ID NO: 1.

In one embodiment, the composition includes about 30 μg/ml of a firstpolypeptide including the amino acid sequence set forth in SEQ ID NO: 1.In one preferred embodiment, the composition includes about 60 μg of afirst polypeptide including the amino acid sequence set forth in SEQ IDNO: 1. In one preferred embodiment, the composition includes about 60 μgof a first polypeptide including the amino acid sequence set forth inSEQ ID NO: 1, wherein the composition preferably has a total volume of0.5 ml. In another embodiment, the composition includes about 120 μg/mlof a first polypeptide including the amino acid sequence set forth inSEQ ID NO: 1.

Second Polypeptide; MNB RLP2086 Subfamily B (B01) Protein

In one embodiment, the composition includes a second polypeptide havingthe amino acid sequence set forth in SEQ ID NO: 2. The polypeptide is afactor H binding protein (fHBP) from N. meningitidis strain CDC1573. Adescription of fHBP is disclosed in WO2012032489 and US patentpublication US 2012/0093852, which are each incorporated by reference intheir entirety. The polypeptide is N-terminally lipidated with threepredominant fatty acids C16:0, C16:1, and C18:1 covalently linked atthree positions of the polypeptide. The second polypeptide includes atotal of 261 amino acids.

The representative primary structure of the MnB rLP2086 B01 protein ispresented in FIG. 5. The primary structure of the protein is illustratedin FIG. 5 using a single letter notation for all amino acids except forthe N-terminal cysteine and glyceryl moieties (illustrated using fullchemical formula). This structure includes the primary structure of theprotein sequence in which the N-terminal cysteine residue is lipidated.The amino group of the N-terminal cysteine residue at the proteinN-terminus is attached to a fatty acid (R1) forming an amide linkage andthe cysteinyl sulfhydryl group is attached to a glycerol moietycontaining two-ester bound fatty acids (R2). The structure of R1 isdeduced to be hexadecanoic acid (C16:0) and the structures of R2 varydepending on the rLP2086 isoforms.

The second polypeptide includes one modification introduced in theN-terminal region for the rLP2086 subfamily B protein, as compared tothe corresponding wild-type sequence from N. meningitidis strainCDC-1573. A glycine in the second position is a consequence ofintroducing a cloning site.

The N-terminal differences from the original Neisserial sequences areshown below.

Comparison of Predicted N-Terminal Sequencesof Recombinant and Neisserial Subfamily B LP2086 ProteinrLP2086 CDC-1573  CGSSGGGGSGGGGVTAD (SEQ ID NO: 24)Neisserial LP2086 CDC-1573  C-SSGGGGSGGGGVTAD (SEQ ID NO: 25)

In one embodiment, the second polypeptide includes a C-G-S-S sequence(SEQ ID NO: 3) at the N-terminus. See the first four amino acid residuesof SEQ ID NO: 2.

>B01  (SEQ ID NO: 2) CGSSGGGGSGGGGVTADIGTGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQEQDPEHSEKMVAKRRFRIGDIAGEHTSFDKLPKDVMATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAVAYIKPDEKHHAVISGSVLYNQDEKGSYSLGIFGEKAQEVAGSAEVETAN GIHHIGLAAKQ

In one embodiment, the second polypeptide includes the amino acidsequence set forth in SEQ ID NO: 2. In one embodiment, the secondpolypeptide has a total of 261 amino acids. In one embodiment, thesecond polypeptide consists of the amino acid sequence set forth in SEQID NO: 2. In another embodiment, the second polypeptide does not furtherinclude a polypeptide having less than 100% sequence identity to SEQ IDNO: 2. In a preferred embodiment, the first polypeptide and the secondpolypeptide includes a C-G-S-S(SEQ ID NO: 3) sequence at the N-terminusof the respective polypeptide.

In a preferred embodiment, the second polypeptide is readily expressedin a recombinant host cell using standard techniques known in the art.In another preferred embodiment, the second polypeptide includes abactericidal epitope on the N- and/or C-domain of SEQ ID NO: 2. In oneembodiment, the second polypeptide includes at least the first 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, or 100 amino acid residues of the amino acid sequenceset forth in SEQ ID NO: 2. Preferably, the second polypeptide includesat least the first 2, more preferably at least the first 4, and mostpreferably, at least the first 8 amino acid residues of SEQ ID NO: 2.

In another embodiment, the second polypeptide includes at least the last4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, or 100 amino acid residues of the amino acidsequence set forth in SEQ ID NO: 2.

In one embodiment, the composition includes about 30 μg/ml of a firstpolypeptide including the amino acid sequence set forth in SEQ ID NO: 2.In one preferred embodiment, the composition includes about 60 μg of afirst polypeptide including the amino acid sequence set forth in SEQ IDNO: 2. In one preferred embodiment, the composition includes about 60 μgof a second polypeptide including the amino acid sequence set forth inSEQ ID NO: 2, wherein the composition preferably has a total volume of0.5 ml. In another embodiment, the composition includes 120 μg/ml of asecond polypeptide including the amino acid sequence set forth in SEQ IDNO: 2.

Saccharides

The term “saccharide” throughout this specification may indicatepolysaccharide or oligosaccharide and includes both. Polysaccharides areisolated from bacteria or isolated from bacteria and sized to somedegree by known methods and optionally by microfluidisation.Polysaccharides can be sized in order to reduce viscosity inpolysaccharide samples and/or to improve filterability for conjugatedproducts. Oligosaccharides have a low number of repeat units (typically5-30 repeat units) and are typically hydrolysed polysaccharides.

Each N. meningitidis capsular saccharide may be conjugated to a carrierprotein independently selected from the group consisting of TT, DT,CRM197, fragment C of TT and protein D. Although one or more N.meningitidis capsular saccharide may be conjugated to different carrierproteins from the others, in one embodiment they are all conjugated tothe same carrier protein. For instance they may all be conjugated to thesame carrier protein selected from the group consisting of TT, DT,CRM197, fragment C of TT and protein D. In this context CRM197 and DTmay be considered to be the same carrier protein as they differ by onlyone amino acid. In a preferred embodiment all the N. meningitidiscapsular saccharides present are conjugated to TT.

If the protein carrier is the same for 2 or more saccharides in thecomposition, the saccharide could be conjugated to the same molecule ofthe protein carrier (carrier molecules having 2 more differentsaccharides conjugated to it) [see for instance WO 04/083251; forexample, a single carrier protein might be conjugated to MenA and MenC;MenA and MenW; MenA and MenY; MenC and MenW; MenC and MenY; Men W andMenY; MenA, MenC and MenW; MenA, MenC and MenY; MenA, MenW and MenY;MenC, MenW and MenY; MenA, MenC, MenW and MenY. Alternatively thesaccharides may each be separately conjugated to different molecules ofthe protein carrier (each molecule of protein carrier only having onetype of saccharide conjugated to it).

In one embodiment, at least 2 different saccharide conjugates areconjugated separately to the same type of carrier protein, wherein oneor more saccharide(s) is/are conjugated to the carrier protein via afirst type of chemical group on the protein carrier, and one or moresaccharide(s) is/are conjugated to the carrier protein via a second(different) type of chemical group on the protein carrier.

In one embodiment the 2 conjugates involve the same saccharide linked tothe same carrier, but by different conjugation chemistries. In analternative embodiment 2 different saccharides are conjugated todifferent groups on the protein carrier.

By “conjugated separately to the same type of carrier protein” it ismeant that the saccharides are conjugated to the same carrierindividually (for example, MenA is conjugated to tetanus toxoid throughan amine group on the tetanus toxoid and MenC is conjugated to tetanustoxoid through a carboxylic acid group on a different molecule oftetanus toxoid.)

The capsular saccharide(s) may be conjugated to the same carrier proteinindependently selected from the group consisting of TT, DT, CRM197,fragment C of TT and protein D. A more complete list of protein carriersthat may be used in the conjugates of the invention is presented below.In this context CRM197 and DT may be considered to be the same carrierprotein as they differ by only one amino acid. In an embodiment all thecapsular saccharides present are conjugated to TT.

The saccharides may include any one of: N. meningitidis serogroup Acapsular saccharide (MenA), N. meningitidis serogroup C capsularsaccharide (MenC), N. meningitidis serogroup Y capsular saccharide(MenY), and N. meningitidis serogroup W capsular saccharide (MenW), orany combination thereof.

The first and second chemical groups present on the protein carrier aredifferent from each other and are ideally natural chemical groups thatmay be readily used for conjugation purposes. They may be selectedindependently from the group consisting of: carboxyl groups, aminogroups, sulphydryl groups, Hydroxyl groups, Imidazolyl groups, Guanidylgroups, and Indolyl groups. In one embodiment the first chemical groupis carboxyl and the second is amino, or vice versa. These groups areexplained in greater detail below.

In a specific embodiment the immunogenic composition comprises at least2 different N. meningitidis capsular saccharides, wherein one or moreis/are selected from a first group consisting of MenA and MenC whichis/are conjugated to the carrier protein via the first type of chemicalgroup on the protein carrier (for instance carboxyl), and one or moredifferent saccharides is/are selected from a second group consisting ofMenC, MenY and MenW which is/are conjugated to the carrier protein viathe second type of chemical group on the protein carrier (for instanceamino).

In a further embodiment the immunogenic composition of the inventioncomprises MenA conjugated via the first type of chemical group (forinstance carboxyl), and MenC conjugated via the second type of chemicalgroup (for instance amino).

In another embodiment the immunogenic composition comprises MenCconjugated via the first type of chemical group (for instance carboxyl),and MenY conjugated via the second type of chemical group (for instanceamino).

In another embodiment the immunogenic composition comprises MenAconjugated via the first type of chemical group (for instance carboxyl),and MenC, MenY and MenW conjugated via the second type of chemical group(for instance amino).

In another embodiment the immunogenic composition comprises MenA andMenC conjugated via the first type of chemical group (for instancecarboxyl), and MenY and MenW conjugated via the second type of chemicalgroup (for instance amino).

The saccharides of the invention included in pharmaceutical(immunogenic) compositions of the invention are conjugated to a carrierprotein such as tetanus toxoid (TT), tetanus toxoid fragment C,non-toxic mutants of tetanus toxin [note all such variants of TT areconsidered to be the same type of carrier protein for the purposes ofthis invention], diphtheria toxoid (DT), CRM197, other non-toxic mutantsof diphtheria toxin [such as CRM176, CRM 197, CRM228, CRM 45 (Uchida etal J. Biol. Chem. 218; 3838-3844, 1973); CRM 9, CRM 45, CRM102, CRM 103and CRM107 and other mutations described by Nicholls and Youle inGenetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc, 1992;deletion or mutation of Glu-148 to Asp, Gln or Ser and/or Ala 158 to Glyand other mutations disclosed in U.S. Pat. Nos. 4,709,017 or 4,950,740;mutation of at least one or more residues Lys 516, Lys 526, Phe 530and/or Lys 534 and other mutations disclosed in U.S. Pat. Nos. 5,917,017or 6,455,673; or fragment disclosed in U.S. Pat. No. 5,843,711] (noteall such variants of DT are considered to be the same type of carrierprotein for the purposes of this invention), pneumococcal pneumolysin(Kuo et al (1995) Infect Immun 63; 2706-13), OMPC (meningococcal outermembrane protein—usually extracted from N. meningitidis serogroupB—EP0372501), synthetic peptides (EP0378881, EP0427347), heat shockproteins (WO 93/17712, WO 94/03208), pertussis proteins (WO 98/58668,EP0471177), cytokines, lymphokines, growth factors or hormones (WO91/01146), artificial proteins comprising multiple human CD4+ T cellepitopes from various pathogen derived antigens (Falugi et al (2001) EurJ Immunol 31; 3816-3824) such as N19 protein (Baraldoi et al (2004)Infect Immun 72; 4884-7) pneumococcal surface protein PspA (WO02/091998), iron uptake proteins (WO 01/72337), toxin A or B of C.difficile (WO 00/61761) or Protein D (EP594610 and WO 00/56360).

In an embodiment, the immunogenic composition of the invention uses thesame type of carrier protein (independently) in at least two, three,four or each of the saccharides contained therein.

In an embodiment, the immunogenic composition of the invention comprisesa N. meningitidis saccharide conjugated to a carrier protein selectedfrom the group consisting of TT, DT, CRM197, fragment C of TT andprotein D.

The immunogenic composition of the invention optionally comprises atleast one meningococcal saccharide (for example MenA; MenC; MenW; MenY;MenA and MenC; MenA and MenW; MenA and MenY; MenC and Men W; Men C andMenY; Men W and MenY; MenA, MenC and MenW; MenA, MenC and MenY; MenA,MenW and MenY; MenC, MenW and MenY or MenA, MenC, MenW and MenY)conjugate having a ratio of Men saccharide to carrier protein of between1:5 and 5:1, between 1:2 and 5:1, between 1:0.5 and 1:2.5 or between1:1.25 and 1:2.5(w/w). In one preferred embodiment, the compositionincludes MenA, MenC, MenW and MenY each conjugated to tetanus toxoid atratios (toxoid to polysaccharide) of about 3, about 3, about 1.5 andabout 1.3, respectively.

The ratio of saccharide to carrier protein (w/w) in a conjugate may bedetermined using the sterilized conjugate. The amount of protein isdetermined using a Lowry assay (for example Lowry et al (1951) J. Biol.Chem. 193, 265-275 or Peterson et al Analytical Biochemistry 100,201-220 (1979)) and the amount of saccharide is determined using ICP-OES(inductively coupled plasma-optical emission spectroscopy) for MenA,DMAP assay for MenC and Resorcinol assay for MenW and MenY (Monsigny etal (1988) Anal. Biochem. 175, 525-530).

In an embodiment, the immunogenic composition of the invention comprisesN. meningitidis saccharide conjugate(s) wherein the N. meningitidissaccharide(s) is conjugated to the carrier protein via a linker, forinstance a bifunctional linker. The linker is optionallyheterobifunctional or homobifunctional, having for example a reactiveamino group and a reactive carboxylic acid group, 2 reactive aminogroups or two reactive carboxylic acid groups. The linker has forexample between 4 and 20, 4 and 12, 5 and 10 carbon atoms. A possiblelinker is ADH. Other linkers include B-propionamido (WO 00/10599),nitrophenyl-ethylamine (Geyer et al (1979) Med. Microbiol. Immunol. 165;171-288), haloalkyl halides (U.S. Pat. No. 4,057,685), glycosidiclinkages (U.S. Pat. Nos. 4,673,574, 4,808,700), hexane diamine and6-aminocaproic acid (U.S. Pat. No. 4,459,286).

The saccharide conjugates present in the immunogenic compositions of theinvention may be prepared by any known coupling technique. Theconjugation method may rely on activation of the saccharide with1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) to form acyanate ester. The activated saccharide may thus be coupled directly orvia a spacer (linker) group to an amino group on the carrier protein.For example, the spacer could be cystamine or cysteamine to give athiolated polysaccharide which could be coupled to the carrier via athioether linkage obtained after reaction with a maleimide-activatedcarrier protein (for example using GMBS) or a holoacetylated carrierprotein (for example using iodoacetimide or N-succinimidylbromoacetatebromoacetate). Optionally, the cyanate ester (optionallymade by CDAP chemistry) is coupled with hexane diamine or ADH and theamino-derivatised saccharide is conjugated to the carrier protein usingusing carbodiimide (e.g. EDAC or EDC) chemistry via a carboxyl group onthe protein carrier. Such conjugates are described in PCT publishedapplication WO 93/15760 Uniformed Services University and WO 95/08348and WO 96/29094.

Other suitable techniques use carbiinides, hydrazides, active esters,norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU.Many are described in WO 98/42721. Conjugation may involve a carbonyllinker which may be formed by reaction of a free hydroxyl group of thesaccharide with CDI (Bethell et al J. Biol. Chem. 1979, 254; 2572-4,Hearn et al J. Chromatogr. 1981. 218; 509-18) followed by reaction ofwith a protein to form a carbamate linkage. This may involve reductionof the anomeric terminus to a primary hydroxyl group, optionalprotection/deprotection of the primary hydroxyl group′ reaction of theprimary hydroxyl group with CDI to form a CDI carbamate intermediate andcoupling the CDI carbamate intermediate with an amino group on aprotein.

The conjugates can also be prepared by direct reductive aminationmethods as described in U.S. Pat. No. 4,365,170 (Jennings) and U.S. Pat.No. 4,673,574 (Anderson). Other methods are described in EP-0-161-188,EP-208375 and EP-0-477508.

A further method involves the coupling of a cyanogen bromide (or CDAP)activated saccharide derivatised with adipic acid hydrazide (ADH) to theprotein carrier by Carbodiimide condensation (Chu C. et al Infect.Immunity, 1983 245 256), for example using EDAC.

In an embodiment, a hydroxyl group (optionally an activated hydroxylgroup for example a hydroxyl group activated by a cyanate ester) on asaccharide is linked to an amino or carboxylic group on a protein eitherdirectly or indirectly (through a linker). Where a linker is present, ahydroxyl group on a saccharide is optionally linked to an amino group ona linker, for example by using CDAP conjugation. A further amino groupin the linker for example ADH) may be conjugated to a carboxylic acidgroup on a protein, for example by using carbodiimide chemistry, forexample by using EDAC. In an embodiment, N. meningitidis capsularsaccharide(s) (or saccharide in general) is conjugated to the linkerfirst before the linker is conjugated to the carrier protein.Alternatively the linker may be conjugated to the carrier beforeconjugation to the saccharide.

In general the following types of chemical groups on a protein carriercan be used for coupling/conjugation:

A) Carboxyl (for instance via aspartic acid or glutamic acid). In oneembodiment this group is linked to amino groups on saccharides directlyor to an amino group on a linker with carbodiimide chemistry e.g. withEDAC.B) Amino group (for instance via lysine). In one embodiment this groupis linked to carboxyl groups on saccharides directly or to a carboxylgroup on a linker with carbodiimide chemistry e.g. with EDAC. In anotherembodiment this group is linked to hydroxyl groups activated with CDAPor CNBr on saccharides directly or to such groups on a linker; tosaccharides or linkers having an aldehyde group; to saccharides orlinkers having a succinimide ester group.C) Sulphydryl (for instance via cysteine). In one embodiment this groupis linked to a bromo or chloro acetylated saccharide or linker withmaleimide chemistry. In one embodiment this group is activated/modifiedwith bis diazobenzidine.D) Hydroxyl group (for instance via tyrosine). In one embodiment thisgroup is activated/modified with bis diazobenzidine.E) Imidazolyl group (for instance via histidine). In one embodiment thisgroup is activated/modified with bis diazobenzidine.F) Guanidyl group (for instance via arginine).G) Indolyl group (for instance via tryptophan).

On a saccharide, in general the following groups can be used for acoupling: OH, COOH or NH2. Aldehyde groups can be generated afterdifferent treatments known in the art such as: periodate, acidhydrolysis, hydrogen peroxide, etc.

Direct Coupling Approaches:

Saccharide-OH+CNBr or CDAP - - - >cyanate ester+NH2-Prot - - ->conjugateSaccharide-aldehyde+NH2-Prot - - - >Schiff base+NaCNBH3 - - - >conjugateSaccharide-COOH+NH2-Prot+EDAC - - - >conjugateSaccharide-NH2+COOH-Prot+EDAC - - - >conjugate

Indirect coupling via spacer (linker) approaches:

Saccharide-OH+CNBr or CDAP - - - >cyanate ester+NH2 - - -NH2 - - - >saccharide - - - NH2+COOH-Prot+EDAC - - - >conjugateSaccharide-OH+CNBr or CDAP - - - >cyanate ester+NH2SH - - - >saccharide - - - SH+SH-Prot (native Protein with an exposedcysteine or obtained after modification of amino groups of the proteinby SPDP for instance) - - - >saccharide-S—S-ProtSaccharide-OH+CNBr or CDAP - - - >cyanate ester+NH2 - - -SH >saccharide - - - SH+maleimide-Prot (modification of aminogroups) - - - >conjugateSaccharide-COOH+EDAC+NH2 - - - NH2 - - - >saccharide - - -NH2+EDAC+COOH-Prot - - - >conjugateSaccharide-COOH+EDAC+NH2 - - - SH - - - >saccharide - - - SH+SH-Prot(native Protein with an exposed cysteine or obtained after modificationof amino groups of the protein by SPDP for instance) - - ->saccharide-S—S-ProtSaccharide-COOH+EDAC+NH2 - - - SH - - - >saccharide - - -SH+maleimide-Prot (modification of amino groups) - - - >conjugateSaccharide-Aldehyde+NH2 - - - NH2 - - - >saccharide - - -NH2+EDAC+COOH-Prot - - - >conjugateNote: instead of EDAC above, any suitable carbodiimide may be used.

In summary, the types of protein carrier chemical group that may begenerally used for coupling with a saccharide are amino groups (forinstance on lysine residues), COOH groups (for instance on aspartic andglutamic acid residues) and SH groups (if accessible) (for instance oncysteine residues).

In an embodiment, at least one of the N. meningitidis capsularsaccharides (or saccharide in general) is directly conjugated to acarrier protein; optionally Men W and/or MenY and/or MenC saccharide(s)is directly conjugated to a carrier protein. For example MenW; MenY;MenC; MenW and MenY; MenW and MenC; MenY and MenC; or MenW, MenY andMenC are directly linked to the carrier protein. Optionally, at leastone of the N. meningitidis capsular saccharides is directly conjugatedby CDAP. For example MenW; MenY; MenC; MenW and MenY; MenW and MenC;MenY and MenC; or MenW, MenY and MenC are directly linked to the carrierprotein by CDAP (see WO 95/08348 and WO 96/29094). In an embodiment, allN. meningitidis capsular saccharides are conjugated to tetanus toxoid.

In an embodiment, the ratio of Men W and/or Y saccharide to carrierprotein is between 1:0.5 and 1:2 (w/w) and/or the ratio of MenCsaccharide to carrier protein is between 1:0.5 and 1:4 or 1:0.5 and1:1.5 (w/w), especially where these saccharides are directly linked tothe protein, optionally using CDAP.

In an embodiment, at least one of the N. meningitidis capsularsaccharide(s) (or saccharide in general) is conjugated to the carrierprotein via a linker, for instance a bifunctional linker. The linker isoptionally heterobifunctional or homobifunctional, having for example areactive amine group and a reative carboxylic acid group, 2 reactiveamine groups or 2 reactive carboxylic acid groups. The linker has forexample between 4 and 20, 4 and 12, 5 and 10 carbon atoms. A possiblelinker is ADH.

In an embodiment, MenA; MenC; or MenA and MenC is conjugated to acarrier protein (for example tetanus toxoid) via a linker.

In an embodiment, at least one N. meningitidis saccharide is conjugatedto a carrier protein via a linker using CDAP and EDAC. For example,MenA; MenC; or MenA and MenC are conjugated to a protein via a linker(for example those with two hydrazino groups at its ends such as ADH)using CDAP and EDAC as described above. For example, CDAP is used toconjugate the saccharide to a linker and EDAC is used to conjugate thelinker to a protein. Optionally the conjugation via a linker results ina ratio of saccharide to carrier protein of between 1:0.5 and 1:6; 1:1and 1:5 or 1:2 and 1:4, for MenA; MenC; or MenA and MenC.

In an embodiment, the MenA capsular saccharide, where present is atleast partially O-acetylated such that at least 50%, 60%, 70%, 80%, 90%,95% or 98% of the repeat units are O-acetylated at at least oneposition. O-acetylation is for example present at least at the O-3position of at least 50%, 60%, 70%, 80%, 90%, 95% or 98% of the repeatunits.

In an embodiment, the MenC capsular saccharide, where present is atleast partially O-acetylated such that at least 30%, 40%, 50%, 60%, 70%,80%, 90%, 95% or 98% of (a2→9)-linked NeuNAc repeat units areO-acetylated at at least one or two positions. O-acetylation is forexample present at the O-7 and/or O-8 position of at least 30%. 40%,50%, 60%, 70%, 80%, 90%, 95% or 98% of the repeat units.

In an embodiment, the MenW capsular saccharide, where present is atleast partially O-acetylated such that at least 30%, 40%, 50%, 60%, 70%,80%, 90%, 95% or 98% of the repeat units are O-acetylated at at leastone or two positions. O-acetylation is for example present at the O-7and/or O-9 position of at least 30%. 40%, 50%, 60%, 70%, 80%, 90%, 95%or 98% of the repeat units.

In an embodiment, the MenY capsular saccharide, where present is atleast partially O-acetylated such that at least 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95% or 98% of the repeat units are O-acetylated at atleast one or two positions. O-acetylation is present at the 7 and/or 9position of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98%of the repeat units.

The percentage of O-acetylation refers to the percentage of the repeatunits containing O-acetylation. This may be measured in the saccharideprior to conjugate and/or after conjugation.

In one embodiment of the invention the immunogenic composition,saccharide present, or each N. meningitidis capsular saccharide present,is conjugated to TT. In a further embodiment each N. meningitidiscapsular saccharide is separately conjugated to a separate carrierprotein. In a further embodiment each N. meningitidis capsularsaccharide conjugate has a saccharide:carrier ratio of 1:5-5:1 or1:1-1:4(w/w). In a further embodiment at least one, two or three N.meningitidis capsular saccharide conjugate(s) is directly conjugated toa carrier protein. In a further embodiment Men W and/or MenY, MenWand/or MenC, MenY and/or MenC, or MenW and MenC and MenY are directlyconjugated to a carrier protein. In a further embodiment at least one,two or three N. meningitidis saccharide conjugate(s) is directlyconjugated by CDAP chemistry. In a further embodiment the ratio of Men Wand/or Y saccharide to carrier protein is between 1:0.5 and 1:2 (w/w).In a further embodiment the ratio of MenC saccharide to carrier proteinis between 1:0.5 and 1:2 (w/w). In a further embodiment at least one,two or three N. meningitidis capsular saccharide(s) are conjugated tothe carrier protein via a linker (which may be bifunctional such ashaving two reactive amino groups (such as ADH) or two reactive carboxylgroups, or a reactive amino group at one end and a reactive carboxylgroup at the other). The linker can have between 4 and 12 carbon atoms.In a further embodiment the or each N. meningitidis capsularsaccharide(s) conjugated via a linker are conjugated to the linker withCDAP chemistry. In a further embodiment the carrier protein isconjugated to the linker using carbodiimide chemistry, for example usingEDAC. In a further embodiment the or each N. meningitidis capsularsaccharide is conjugated to the linker before the carrier protein isconjugated to the linker. In a further embodiment MenA is conjugated toa carrier protein via a linker (the ratio of MenA saccharide to carrierprotein may be between 1:2 and 1:5 (w/w)). In a further embodiment MenCis conjugated to a carrier protein via a linker (the ratio of MenCsaccharide to carrier protein may be between 1:2 and 1:5 (w/w)).

By using native or slightly sized polysaccharide conjugates, one or moreof the following advantages may be realised: 1) a conjugate having highimmungenicity which is filterable through a 0.2 micron filter; 2) immunememory may be enhanced (as in example three); 3) the alteration of theratio of polysaccharide to protein in the conjugate such that the ratioof polysaccharide to protein (w/w) in the conjugate may be increased(this can result in a reduction of the carrier suppression effect); 4)immunogenic conjugates prone to hydrolysis (such as MenA conjugates) maybe stabilised by the use of larger polysaccharides for conjugation. Theuse of larger polysaccharides can result in more cross-linking with theconjugate carrier and may lessen the liberation of free saccharide fromthe conjugate. The conjugate vaccines described in the prior art tend todepolymerise the polysaccharides prior to conjugation in order toimprove conjugation. Meningococcal (or saccharide) conjugate vaccinesretaining a larger size of saccharide can provide a good immune responseagainst meningococcal disease.

The immunogenic composition of the invention may thus comprise one ormore saccharide conjugates wherein the average size of each saccharidebefore conjugation is above 50 kDa, 75 kDa, 100 kDa, 110 kDa, 120 kDa or130 kDa. In one embodiment the conjugate post conjugation should bereadily filterable through a 0.2 micron filter such that a yield of morethan 50, 60, 70, 80, 90 or 95% is obtained post filtration compared withthe pre filtration sample.

In particular, the immunogenic composition of the invention comprises N.meningitidis capsular saccharides from at least one, two, three or fourof serogroups A, C, W and Y conjugated to a carrier protein, wherein theaverage size (weight-average molecular weight; Mw) of at least one, two,three or four or each N. meningitidis saccharide is above 50 kDa, 60kDa, 75 kDa, 100 kDa, 110 kDa, 120 kDa or 130 kDa.

In a preferred embodiment, the average Mw of the MenA_(AH)-TT conjugateis at least 250 kDa, 260 kDa, 270 kDa, 280 kDa, or 290 kDa, mostpreferably about 300 kDa, and at most 350 kDa or 330 kDa. In a preferredembodiment, the average Mw of the MenC_(AH)-TT conjugate is at least 150kDa, 160 kDa, 170 kDa, 180 kDa, or 190 kDa, most preferably about 200kDa, and at most 250 kDa or 230 kDa. In a preferred embodiment, theaverage Mw of the MenW-TT conjugate is at least 240, 250 kDa, 260 kDa,or 270 kDa, most preferably about 280 kDa, and at most 330 kDa or 310kDa. In a preferred embodiment, the average Mw of the MenY-TT conjugateis at least 220 kDa, 230 kDa, 240 kDa, or 250 kDa, most preferably about270 kDa, and at most 320 kDa or 300 kDa.

The immunogenic composition may comprise N. meningitidis capsularsaccharides from at least one, two, three or four of serogroups A, C, Wand Y conjugated to a carrier protein, wherein at least one, two, threeor four or each N. meningitidis saccharide is either a native saccharideor is sized by a factor up to ×2, ×3, ×4, ×5, ×6, ×7, ×8, ×9 or ×10relative to the weight average molecular weight of the nativepolysaccharide.

For the purposes of the invention, “native polysaccharide” refers to asaccharide that has not been subjected to a process, the purpose ofwhich is to reduce the size of the saccharide. A polysaccharide canbecome slightly reduced in size during normal purification procedures.Such a saccharide is still native. Only if the polysaccharide has beensubjected to sizing techniques would the polysaccharide not beconsidered native.

For the purposes of the invention, “sized by a factor up to ×2” meansthat the saccharide is subject to a process intended to reduce the sizeof the saccharide but to retain a size more than half the size of thenative polysaccharide. ×3, ×4 etc. are to be interpreted in the same wayi.e. the saccharide is subject to a process intended to reduce the sizeof the polysaccharide but to retain a size more than a third, a quarteretc. the size of the native polysaccharide.

In an aspect of the invention, the immunogenic composition comprises N.meningitidis capsular saccharides from at least one, two, three or fourof serogroups A, C, W and Y conjugated to a carrier protein, wherein atleast one, two, three or four or each N. meningitidis saccharide isnative polysaccharide.

In an aspect of the invention, the immunogenic composition comprises N.meningitidis capsular saccharides from at least one, two, three or fourof serogroups A, C, W and Y conjugated to a carrier protein, wherein atleast one, two, three or four or each N. meningitidis saccharide issized by a factor up to ×1.5, ×2, ×3, ×4, ×5, ×6, ×7, ×8, ×9 or ×10.

The immunogenic compositions of the invention optionally compriseconjugates of: N. meningitidis serogroup C capsular saccharide (MenC),serogroup A capsular saccharide (MenA), serogroup W135 capsularsaccharide (MenW), serogroup Y capsular saccharide (MenY), serogroup Cand Y capsular saccharides (MenCY), serogroup C and A capsularsaccharides (MenAC), serogroup C and W capsular saccharides (MenCW),serogroup A and Y capsular saccharide (MenAY), serogroup A and Wcapsular saccharides (MenAVV), serogroup W and Y capsular saccharides(Men WY), serogroup A, C and W capsular saccharide (MenACW), serogroupA, C and Y capsular saccharides (MenACY); serogroup A, W135 and Ycapsular saccharides (MenAWY), serogroup C, W135 and Y capsularsaccharides (MenCWY); or serogroup A, C, W135 and Y capsular saccharides(MenACWY). This is the definition of “one, two, three or four”, or “atleast one of” of serogroups A, C, W and Y, or of each N. meningitidissaccharide where mentioned herein.

In an embodiment, the average size of at least one, two, three, four oreach N. meningitidis saccharide is between 50 KDa and 1500 kDa, 50 kDaand 500 kDa, 50 kDa and 300 KDa, 101 kDa and 1500 kDa, 101 kDa and 500kDa, 101 kDa and 300 kDa as determined by MALLS.

In an embodiment, the MenA saccharide, where present, has a molecularweight of 50-500 kDa, 50-100 kDa, 100-500 kDa, 55-90 KDa, 60-70 kDa or70-80 kDa or 60-80 kDa.

In an embodiment, the MenC saccharide, where present, has a molecularweight of 100-200 kDa, 50-100 kDa, 100-150 kDa, 101-130 kDa, 150-210 kDaor 180-210 kDa.

In an embodiment the MenY saccharide, where present, has a molecularweight of 60-190 kDa, 70-180 kDa, 80-170 kDa, 90-160 kDa, 100-150 kDa or110-140 kDa, 50-100 kDa, 100-140 kDa, 140-170 kDa or 150-160 kDa.

In an embodiment the MenW saccharide, where present, has a molecularweight of 60-190 kDa, 70-180 kDa, 80-170 kDa, 90-160 kDa, 100-150 kDa,110-140 kDa, 50-100 kDa or 120-140 kDa.

The molecular weight or average molecular weight of a saccharide hereinrefers to the weight-average molecular weight (Mw) of the saccharidemeasured prior to conjugation and is measured by MALLS.

The MALLS technique is well known in the art and is typically carriedout as described in example 2. For MALLS analysis of meningococcalsaccharides, two columns (TSKG6000 and 5000 PWxl) may be used incombination and the saccharides are eluted in water. Saccharides aredetected using a light scattering detector (for instance Wyatt Dawn DSPequipped with a 10 mW argon laser at 488 nm) and an inferometricrefractometer (for instance Wyatt Otilab DSP equipped with a P100 celland a red filter at 498 nm).

In an embodiment the N. meningitidis saccharides are nativepolysaccharides or native polysaccharides which have reduced in sizeduring a normal extraction process.

In an embodiment, the N. meningitidis saccharides are sized bymechanical cleavage, for instance by microfluidisation or sonication.Microfluidisation and sonication have the advantage of decreasing thesize of the larger native polysaccharides sufficiently to provide afilterable conjugate (fro example through a 0.2 micron filter). Sizingis by a factor of no more than ×20, ×10, ×8, ×6, ×5, ×4, ×3, ×2 or ×1.5.

In an embodiment, the immunogenic composition comprises N. meningitidisconjugates that are made from a mixture of native polysaccharides andsaccharides that are sized by a factor of no more than ×20. For example,saccharides from MenC and/or MenA are native. For example, saccharidesfrom MenY and/or MenW are sized by a factor of no more than ×20, ×10,×8, ×6, ×5, ×4, ×3 or ×2. For example, an immunogenic compositioncontains a conjugate made from MenY and/or MenW and/or MenC and/or MenAwhich is sized by a factor of no more then ×10 and/or is microfluidised.For example, an immunogenic composition contains a conjugate made fromnative MenA and/or MenC and/or MenW and/or MenY. For example, animmunogenic composition comprises a conjugate made from native MenC. Forexample, an immunogenic composition comprises a conjugate made fromnative MenC and MenA which is sized by a factor of no more then ×10and/or is microfluidised. For example, an immunogenic compositioncomprises a conjugate made from native MenC and MenY which is sized by afactor of no more then ×10 and/or is microfluidised.

In an embodiment, the polydispersity of the saccharide is 1-1.5, 1-1.3,1-1.2, 1-1.1 or 1-1.05 and after conjugation to a carrier protein, thepolydispersity of the conjugate is 1.0-2.5, 1.0-2.0. 1.0-1.5, 1.0-1.2,1.5-2.5, 1.7-2.2 or 1.5-2.0. All polydispersity measurements are byMALLS.

Saccharides are optionally sized up to 1.5, 2, 4, 6, 8, 10, 12, 14, 16,18 or 20 times from the size of the polysaccharide isolated frombacteria.

In one embodiment each N. meningitidis saccharide is either a nativepolysaccharide or is sized by a factor of no more than ×10. In a furtherembodiment each N. meningitidis capsular saccharide is a nativepolysaccharide. In a further embodiment at least one, two, three or fourN. meningitidis capsular saccharide(s) is sized by microfluidization. Ina further embodiment each N. meningitidis capsular saccharide is sizedby a factor of no more than ×10. In a further embodiment the N.meningitidis conjugates are made from a mixture of nativepolysaccharides and saccharides that are sized by a factor of no morethan ×10. In a further embodiment the capsular saccharide from serogroupY is sized by a factor of no more than ×10. In a further embodimentcapsular saccharides from serogroups A and C are native polysaccharidesand saccharides from serogroups W135 and Y are sized by a factor of nomore than ×10. In a further embodiment the average size of each N.meningitidis capular saccharide is between 50 kDa and 300 KDa or 50 kDaand 200 kDa. In a further embodiment the immunogenic compositioncomprises a MenA capsular saccharide having an average size of above 50kDa, 75 kDa, 100 kDa or an average size of between 50-100 kDa or 55-90KDa or 60-80 kDa. In a further embodiment the immunogenic compositioncomprises a MenC capsular saccharide having an average size of above 50kDa, 75 kDa, 100 kDa or between 100-200 kDa, 100-150 kDa, 80-120 kDa,90-110 kDa, 150-200 kDa, 120-240 kDa, 140-220 kDa, 160-200 kDa or190-200 kDa. In a further embodiment the immunogenic compositioncomprises a MenY capsular saccharide, having an average size of above 50kDa, 75 kDa, 100 kDa or between 60-190 kDa or 70-180 kDa or 80-170 kDaor 90-160 kDa or 100-150 kDa, 110-145 kDa or 120-140 kDa. In a furtherembodiment the immunogenic composition comprises a MenW capsularsaccharide having an average size of above 50 kDa, 75 kDa, 100 kDa orbetween 60-190 kDa or 70-180 kDa or 80-170 kDa or 90-160 kDa or 100-150kDa, 140-180 kDa, 150-170 kDa or 110-140 kDa.

In an embodiment of the invention, the saccharide dose of each of the atleast two, three, four or each of the N. meningitidis saccharideconjugates is optionally the same, or approximately the same.

In an embodiment, the immunogenic composition of the invention isadjusted to or buffered at, or adjusted to between pH 7.0 and 8.0, pH7.2 and 7.6 or around or exactly pH 7.4.

The immunogenic composition or vaccines of the invention are optionallylyophilised in the presence of a stabilising agent for example a polyolsuch as sucrose or trehalose.

For the N. meningitidis saccharide combinations discussed above, it maybe advantageous not to use any aluminium salt adjuvant or any adjuvantat all.

The active agent can be present in varying concentrations in thepharmaceutical composition or vaccine of the invention. Typically, theminimum concentration of the substance is an amount necessary to achieveits intended use, while the maximum concentration is the maximum amountthat will remain in solution or homogeneously suspended within theinitial mixture. For instance, the minimum amount of a therapeutic agentis optionally one which will provide a single therapeutically effectivedosage. For bioactive substances, the minimum concentration is an amountnecessary for bioactivity upon reconstitution and the maximumconcentration is at the point at which a homogeneous suspension cannotbe maintained.

In another embodiment, the composition includes a conjugate of aNeisseria meningitidis serogroup X capsular polysaccharide and a carriermolecule. The structure of the group X capsular polysaccharide consistsof N-acetylglucosamine-4-phosphate residues held together by al-4phosphodiester bonds without O-acetyl groups. The carrier molecule maybe a diphtheria or tetanus toxoid, CRM 197 or protein D. In a preferredembodiment, as exemplified in the Examples, the composition does notinclude a conjugate of a N. meningitidis serogroup X capsularpolysaccharide.

Stability

The terms “stable” and “stability” refer the ability of an antigen toremain immunogenic over a period of time. Stability may be measured inpotency over time. The terms “stable” and “stability” further refer tothe physical, chemical, and conformational stability of the immunogeniccomposition. Instability of a protein composition may be caused bychemical degradation or aggregation of the protein molecules to formhigher order polymers, by dissociation of the heterodimers intomonomers, deglycosylation, modification of glycosylation, or any otherstructural modification that reduces at least one biological activity ofthe protein composition included in the present invention. Stability maybe assessed by methods well-known in the art, including measurement of asample's light scattering, apparent attenuation of light (absorbance, oroptical density), size (e.g. by size exclusion chromatography), in vitroor in vivo biological activity and/or properties by differentialscanning calorimetry (DSC). Other methods for assessing stability areknown in the art and can also be used according to the presentinvention.

In some embodiments, an antigen in a stable formulation of the inventionmay maintain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% potency, as compared to a reference standard, for atleast 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9months, 12 months, 18 months, 24 months, 30 months, 36 months, 42months, 48 months, 54 months, or 60 months. In some embodiments, anantigen in a stable formulation of the invention may maintain at least50% potency, as compared to a reference standard, for at least 1 year, 2years, 3 years, 4 years or 5 years. The terms “stable” and “stability”also refer to the ability of an antigen to maintain epitopes orimmunoreactivity over a period of time. For example, an antigen in astable formulation of the invention may maintain at least 50%, 60%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of its epitopes orimmunoreactivity, as compared to a reference standard, for at least 1month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 12months, 18 months, 24 months, 30 months, 36 months, 42 months, 48months, 54 months, or 60 months. In some embodiments, stability ismeasured with respect to an environmental condition. Non-limitingexamples of environmental conditions include light, temperature,freeze/thaw cycles, agitation, and pH. One of skill in the art would beable to determine the presence of antigenic epitopes or immunoreactivityusing the methods disclosed herein or other methods known in the art. Insome embodiments, the stability of an antigen is measured from the dateof its formulation. In some embodiments, the stability of an antigen ismeasured from the date of a change in its storage conditions.Non-limiting examples of changes in storage conditions include changingfrom frozen to refrigerated, changing from frozen to room temperature,changing from refrigerated to room temperature, changing fromrefrigerated to frozen, changing from room temperature to frozen,changing from room temperature to refrigerated, changing from light todark, or introduction of agitation.

In one embodiment, the terms “stable” and “stability” includes theability of an antigen to be bound to aluminum. For example, a stableformulation of the invention includes at least 50%, 60%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of a protein that is bound toaluminum (e.g., aluminum phosphate) in the formulation, as compared to areference standard, for at least 1 hour, 6 hours, 12 hours, 18 hours, 24hours, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9months, 12 months, 18 months, 24 months, 30 months, 36 months, 42months, 48 months, 54 months, or 60 months. See, for example Example 13.In a preferred embodiment, at least 90%, more preferably at least 95%,and most preferably at least 99% of the total Subfamily A rLP2086polypeptide (e.g., a polypeptide that includes the amino acid sequenceset forth in SEQ ID NO: 1) is bound to aluminum in the composition. In apreferred embodiment, at least 90%, more preferably at least 95%, andmost preferably at least 99% of the total Subfamily B rLP2086polypeptide (e.g., a polypeptide that includes the amino acid sequenceset forth in SEQ ID NO: 2) is bound to aluminum in the composition.

Determination of Aluminum Binding. A composition comprising aluminum andat least one protein antigen was centrifuged such that the aluminum waspelleted. Centrifugation of aluminum absorbed proteins is known in theart. See e.g., Egan et al., Vaccine, Vol. 27(24): 3175-3180 (2009).Aluminum-bound protein was also pelleted, while non-aluminum-boundprotein remained in the supernatant. Total protein in the supernatantand pellet were determined by Lowry Assay. The percentage bound proteinwas calculated by dividing the total protein in the supernatant by thetotal protein added to the composition and multiplying by 100%.Similarly, the percentage unbound protein was calculated by dividing thetotal protein in the supernatant by the total protein added to thecomposition and multiplying by 100%. For compositions comprising bothSubfamily A and Subfamily B antigens, the individual Subfamily A and Bprotein concentrations in the supernatant were determined byion-exchange chromatography. The separation and elution of Subfamily Aand B proteins was carried out using a strong anion column and a highsalt concentration eluent. Both Subfamily A and B proteins were detectedand quantified using a fluorescence detector set at Excitation=280 runand Emission=310 run. Subfamily A and Subfamily B proteins elute atdistinct retention times and were quantified using a standard curvegenerated against a rLP2086 protein reference material. The percentageunbound protein was calculated by dividing the total protein in thesupernatant by the total protein added to the composition andmultiplying by 100%. The percentage bound protein was calculated bysubtracting the percentage unbound protein from 100%.

Polysorbate-80

Polysorbate 80 (PS-80) is a non-ionic surfactant. Accelerated stabilitystudies using an in vitro monoclonal antibody based potency assaydemonstrated instability of the subfamily B protein at higher molarratios of PS-80 to MnB rLP2086 protein in the final formulation. Furtherexperiments with varying ratios of PS-80 have demonstrated that theoptimal molar ratio of PS-80 to MnB rLP2086 protein is approximately2.8±1.4 to retain potency.

The concentration of PS-80 in the composition is dependent on a molarratio of PS-80 to the polypeptide. In one embodiment, the compositionincludes a 2.8±1.4 molar ratio of PS-80 to the first polypeptide and tothe second polypeptide. In one embodiment, the composition includes a2.8±1.1 molar ratio of PS-80 to the first polypeptide and to the secondpolypeptide. In one embodiment, the composition includes at least 1.9,2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, or 3.3molar ratio of PS-80 to polypeptide. In one embodiment, the compositionincludes at most 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0,or 2.9 molar ratio of PS-80 to polypeptide. Any minimum value may becombined with any maximum value described herein to define a range.Preferably, the composition includes a 2.8 molar ratio of PS-80 topolypeptide.

The PS-80 to polypeptide molar ratio is determined by calculation fromthe measured concentration of PS-80 and the measured total polypeptideconcentration, in which both values are expressed in moles. For example,PS-80 to Protein molar ratio is determined by calculation of themeasured concentration of PS-80 (e.g., by reverse phase high pressureliquid chromatography (RP-HPLC)) to the measured total proteinconcentration (e.g., by ion exchange-high pressure liquid chromatography(IEX-HPLC)) in the final drug substance, where both values are expressedin moles.

A RP-HPLC is used to quantitate the concentration of Polysorbate 80 invaccine formulations. The concentration of detergent is determined bysaponification of the fatty acid moiety; Polysorbate 80 is converted tofree oleic acid by alkaline hydrolysis at 40° C. The sample is separatedby RP-HPLC using a C18 column and quantitated using a UV detector at awavelength of 200 nm.

The first and the second polypeptides are resolved by anion-exchangeHPLC. rLP2086(fHBP) Subfamily A and B proteins elute at distinctretention times and are quantitated using a standard curve generatedagainst the respective rLP2086 protein reference material.

The term “molar ratio” and a description of an immunogenic compositionincluding a fHBP and PS-80 is further disclosed in WO2012025873 and USpatent publication US 2013/0171194, which are each incorporated byreference in their entirety.

The term “molar ratio” as used herein refers to the ratio of the numberof moles of two different elements in a composition. In someembodiments, the molar ratio is the ratio of moles of detergent to molesof polypeptide. In some embodiments, the molar ratio is the ratio ofmoles of PS-80 to moles of protein. In one embodiment, based on theprotein and Polysorbate 80 concentrations, the Molar Ratio may becalculated using the following equation:

${{Molar}\mspace{14mu} {Ratio}} = {\frac{\% \mspace{14mu} {PS}\text{-}80}{{mg}\text{/}{ml}\mspace{14mu} {protein}} \times 216}$

In one embodiment, the composition includes a molar ratio of PS-80 toMnB rLP2086 protein between 1.4 to 4.2 to retain potency. In oneembodiment, the composition includes at least 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, or 2.8. In oneembodiment, the composition includes at most 4.2, 4.1, 4.0, 3.9, 3.8,3.7, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, or 2.8. Any minimumvalue may be combined with any maximum value described herein to definea range.

In one embodiment, the composition includes about 0.0015, 0.0017,0.0019, 0.0021, 0.0023, 0.0025, 0.0027, 0.0029, 0.0031, 0.0033, 0.0035,0.0037, 0.0039, 0.0041, 0.0043, 0.0045, 0.0047, 0.0049, 0.0051 mg/mLPS-80. Preferably, the composition includes about 0.0035 mg/mL PS-80.

In another embodiment, the composition includes at least 10 μg, 11 μg,12 μg, 13 μg, 14 μg, 15 μg, 16 μg, 17 μg, 18 μg, 19 μg, 20 μg, 21 μg, 22μg, 23 μg, 24 μg, or 25 μg PS-80. In another embodiment, the compositionincludes at most 30 μg, 29 μg, 28 μg, 27 μg, 26 μg, 25 μg, 24 μg, 23 μg,22 μg, 21 μg, 20 μg, 19 μg, or 18 μg PS-80. Any minimum value may becombined with any maximum value described herein to define a range. In apreferred embodiment, the composition includes at least 10 μg and atmost 20 μg PS-80. In a most preferred embodiment, the compositionincludes about 18 μg PS-80.

In another embodiment, the composition includes a PS-80 concentrationranging from 0.0005% to 1%. For example, the PS-80 concentration in thecomposition may be at least 0.0005%, 0.005%, 0.01%, 0.02%, 0.03%, 0.04%,0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%,0.7%, 0.8%, 0.9%, 1%, or 1.1% PS-80. In one embodiment, the PS-80concentration in the composition may be at most 2.0%, 1.9%, 1.8%, 1.7%,1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.9%, 0.8%, or 0.7% PS-80. Ina preferred embodiment, the composition includes about 0.07% PS-80. Anyminimum value may be combined with any maximum value described herein todefine a range.

The inventors surprisingly discovered that while a composition thatincludes a combination of the first composition and the secondcomposition may have a different molar ratio of polysorbate-80 inrelation to the MnB rLP2086 polypeptides, as compared to the molar ratioof polysorbate-80 in relation to the MnB rLP2086 polypeptides in thefirst composition, additional surfactant for the combined compositionwas surprisingly not necessary to maintain solubility and stability ofthe MnB rLP2086 polypeptides in the combined composition. Accordingly,in one embodiment, the kit does not comprise greater than 0.02 mgpolysorbate-80.

Aluminum

The composition includes aluminum as aluminum phosphate. AlPO₄ is addedas a stabilizer to provide enhanced manufacturability and stability. Theprocess for producing an aluminum phosphate is described in US patentpublication US 2009/0016946, which is incorporated by reference in itsentirety. In one embodiment, the composition does not further include amultivalent cation, other than aluminum. In one embodiment, thecomposition does not further include Al(OH)₃ or Al(SO₄)₃.

In one embodiment, the composition includes at least 50 μg, 60 μg, 70μg, 80 μg, 90 μg, 100 μg, 110 μg, 120 μg, 130 μg, 140 μg, 150 μg, 160μg, 170 μg, 180 μg, 190 μg, 200 μg, 210 μg, 220 μg, 230 μg, 240 μg, or250 μg aluminum. In one embodiment, the composition includes at most 500μg, 490 μg, 480 μg, 470 μg, 460 μg, 450 μg, 440 μg, 430 μg, 420 μg, 410μg, 400 μg, 390 μg, 380 μg, 370 μg, 360 μg, 350 μg, 340 μg, 330 μg, 320μg, 310 μg, 300 μg, 290 μg, 280 μg, 270 μg, 260 μg, or 250 μg aluminum.Any minimum value may be combined with any maximum value describedherein to define a range. In a most preferred embodiment, thecomposition includes 250 μg aluminum.

In one embodiment, the composition includes at least 0.005 mg/ml, 0.01mg/ml, 0.02 mg/ml, 0.03 mg/ml, 0.04 mg/ml, 0.05 mg/ml, 0.06 mg/ml, 0.07mg/ml, 0.08 mg/ml, 0.09 mg/ml, 0.10 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4mg/ml, or 0.5 mg/ml aluminum phosphate. In one embodiment, thecomposition includes at most 2.0 mg/ml, 1.9 mg/ml, 1.8 mg/ml, 1.7 mg/ml,1.6 mg/ml, 1.5 mg/ml, 1.4 mg/ml, 1.3 mg/ml, 1.2 mg/ml, 1.1 mg/ml, 1.0mg/ml, 0.9 mg/ml, 0.8 mg/ml, or 0.7 mg/ml PS-80. In a preferredembodiment, the composition includes about 0.07 mg/ml PS-80. Any minimumvalue may be combined with any maximum value described herein to definea range. In a preferred embodiment, the composition includes 0.5 mg/mlaluminum phosphate. In a most preferred embodiment, the compositionincludes 0.5 mg aluminum/ml as aluminum phosphate (AlPO₄). Thisconcentration maintains binding (at least 90% binding or better) of thesubfamily A and B proteins to aluminum.

The inventors surprisingly discovered that while a composition that acombination of the first composition and the second composition couldchange the percentage of MnB rLP2086 polypeptides bound to the aluminum,when compared to the percentage of MnB rLP2086 polypeptides bound to thealuminum in the first composition, the combination of the first andsecond compositions surprisingly maintained binding of at least 90% ofthe total MnB rLP2086 polypeptides to the aluminum. Accordingly, in oneembodiment, the percentage of total MnB rLP2086 polypeptides to thealuminum in the combined composition is at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%. Preferably, the percentage of total MnB rLP2086polypeptides to the aluminum in the combined composition is at least90%, more preferably at least 95%, and most preferably at least 100%.

In another embodiment, the concentration of polypeptides bound to thealuminum in the immunogenic composition is not decreased after 24 hours,as compared to the concentration of polypeptides bound to the aluminumin the liquid composition prior to reconstituting the lyophilizedcomposition. In another embodiment, the concentration of MenA_(AH)-TTconjugate in the immunogenic composition is not decreased after 24hours, as compared to the concentration of the MenA_(AH)-TT conjugate inthe lyophilized composition. In one embodiment, the concentration isdecreased by at most 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% after 24 hours, as compared tothe respective concentration in the liquid composition prior toreconsititution.

In another embodiment, the concentration of MenC_(AH)-TT conjugate inthe immunogenic composition is not decreased after 24 hours, as comparedto the concentration of the MenC_(AH)-TT conjugate in the lyophilizedcomposition. In another embodiment, the concentration of MenW-TTconjugate in the immunogenic composition is not decreased after 24hours, as compared to the concentration of the MenW-TT conjugate in thelyophilized composition. In another embodiment, the concentration ofMenY-TT conjugate in the immunogenic composition is not decreased after24 hours, as compared to the concentration of the MenY-TT conjugate inthe lyophilized composition. In one embodiment, the concentration isdecreased by at most 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% after 24 hours, as compared tothe respective concentration in the lyophilized composition prior toreconstitution.

Excipients

In one embodiment, the composition includes histidine. In oneembodiment, the composition includes at least 650 μg, 660 μg, 670 μg,680 μg, 690 μg, 700 μg, 710 μg, 720 μg, 730 μg, 740 μg, 750 μg, 760 μg,770 μg, 780 μg, 790 μg, 800 μg, 810 μg, 820 μg, 830 μg, 840 μg, or 850μg of histidine. In one embodiment, the composition includes at most1560 μg, 1500 μg, 1400 μg, 1300 μg, 1200 μg, 1100 μg, 1000 μg, 950 μg,900 μg, 890 μg, 880 μg, 870 μg, 860 μg, 850 μg, 840 μg, 830 μg, 820 μg,810 μg, 800 μg, 790 μg, or 780 μg of histidine. Any minimum value may becombined with any maximum value described herein to define a range.Preferably, the composition includes 780 μg histidine.

In one embodiment, the composition includes a tris, phosphate, orsuccinate buffer. In a preferred embodiment, the composition does notinclude tris buffer. In a preferred, the composition does not includephosphate buffer. In one preferred embodiment, the composition does notinclude succinate buffer. In a preferred embodiment, the compositionincludes histidine buffer.

In one embodiment, the composition includes sodium chloride. Sodiumchloride concentration in MenABCWY composition may vary between160.5-161.1 mM.

In one embodiment, the pH of the composition is between 5.5 and 7.5. Ina preferred embodiment, the pH of the composition is between 5.8 and7.0, most preferably pH 5.8 to pH 6.0. In one embodiment, the pH of thecomposition is at most 6.1. In one embodiment, the pH of the compositionis 5.8.

Kits

A further aspect of the invention is a kit for administering a dose of acomposition for eliciting bactericidal antibodies against Neisseriameningitidis in a mammal.

In one aspect, the kit includes a first composition including a firstpolypeptide as described above and a second polypeptide as describedabove. In a preferred embodiment, the first polypeptide comprises theamino acid sequence set forth in SEQ ID NO: 1. In another preferredembodiment, the second polypeptide comprises the amino acid sequence setforth in SEQ ID NO: 2. The kit further includes a second compositionincluding a MenA_(AH)-TT conjugate, a MenC_(AH)-TT conjugate, a MenW-TTconjugate, and a MenY-TT conjugate. In one embodiment, the kit includesat least two containers, wherein a first container includes the firstcomposition, a second container includes the second composition.

In one embodiment, the kit includes a liquid first composition and alyophilized second composition. Preferably, the kit includes a liquidMnB bivalent rLP2086 composition and a lyophilized MenACWY-TTcomposition.

The inventors surprisingly discovered that while a composition thatincludes a combination of the first composition and the secondcomposition changes the molar ratio of polysorbate-80 in relation to theMnB rLP2086 polypeptides in the first composition, additional surfactantfor the combined composition was surprisingly not necessary to maintainsolubility and stability of the MnB rLP2086 polypeptides in the combinedcomposition. Accordingly, in one embodiment, the kit does not comprisegreater than 0.02 mg polysorbate-80.

In one embodiment of the invention, the kit does not further compriseany one of the following commercial immunogenic compositions: MENACTRA®,MENVEO®, ADACEL®, HAVRIX®, GARDASIL®, REPEVAX, or any combinationthereof. For example, the kit preferably does not further include ameningococcal A, C, Y and W-135 polysaccharide conjugate (MCV4)composition, wherein the carrier protein is diphtheria toxoid. In oneembodiment, the kit does not further include a meningococcal A, C, Y andW-135 polysaccharide conjugate (MCV4) composition, wherein the carrierprotein is CRM197. In one embodiment, the kit does not further compriseNIMENRIX vaccine, wherein NIMENRIX comprises a diluent consisting ofsodium chloride and water.

Bactericidal Activity

Disease incidence of MnB is approximately 1 in 100,000, meaning thatextremely large numbers of subjects (400,000 to over 6 million) would berequired to support a statistically significant assessment of efficacy.Thus, a serum bactericidal assay using human complement (hSBA), which isa surrogate of protection and vaccine efficacy, is used to assessimmunogenicity in clinical trials.

Pfizer has built an extensive MnB strain collection (N=at least 1263)comprising IMD-causing isolates from Years 2000 to 2006. The MnBisolates were systematically collected from the US Centers for DiseaseControl and Prevention (CDC) and health and reference laboratories fromEuropean countries.

In one embodiment, immune response induced by administering thecomposition to a human is determined using a serum bactericidal assayusing human complement (hSBA) against four N. meningitidis serogroup B(MnB) strains. The MnB strains used in the hSBA were selected from thestrain pool. The strain pool represented a collection of systematicallycollected clinically relevant N. meningitidis strains.

The high proportion of hSBA response to all test strains, especiallystrains expressing lipoprotein 2086 variants with sequences heterologousto both the first polypeptide and the second polypeptide suggests thatthe composition is a broadly protective vaccine are sufficient to conferhigh seroprotection against N. meningitidis strains expressing rLP2086(FHBP) from at least serogroup B, including additional serogroups, suchas serogroup X.

Subfamily A Strains

In one embodiment, the hSBA strain is an N. meningitidis strain thatexpresses LP2086 (fHBP) subfamily A protein. In one embodiment, the hSBAstrain is an LP2086 (fHBP) subfamily A strain that expresses alipoprotein 2086 variant that is heterologous to a N. meningitidisstrain expressing A05. For example, in one embodiment, the hSBA strainis an LP2086 (fHBP) subfamily A strain that expresses a lipoprotein 2086variant that is heterologous to strain M98250771.

In one embodiment, the hSBA strain is a N. meningitidis strainexpressing fHBP A10. In one embodiment, the hSBA strain is a N.meningitidis strain expressing LP2086 (fHBP) A22. In one embodiment, thehSBA strain is a N. meningitidis strain expressing LP2086 (fHBP) A56. Ina further embodiment, the hSBA strains are LP2086 (fHBP) A22 and LP2086(fHBP) A56 strains. In another embodiment, the hSBA strain is a N.meningitidis strain expressing LP2086 A04. In one embodiment, the hSBAstrain is a N. meningitidis strain expressing LP2086 A05. In oneembodiment, the hSBA strain is a N. meningitidis strain expressingLP2086 A12. In one embodiment, the hSBA strain is a N. meningitidisstrain expressing LP2086 A22. In one embodiment, the hSBA strain is a N.meningitidis strain expressing LP2086 A12. In one embodiment, the hSBAstrain is a N. meningitidis strain expressing LP2086 A04. In oneembodiment, the hSBA strain is a N. meningitidis strain expressingLP2086 A19. In one embodiment, the hSBA strain is a N. meningitidisstrain expressing LP2086 A07. In a further embodiment, the hSBA strainincludes any one of an A22-, A12-, A19-, A05-, and A07-expressingstrain. In one embodiment, the hSBA strains include any one of an A06-,A15-, and A29-expressing strain.

In one embodiment, the immune response is bactericidal against a N.meningitidis serogroup B fHPB subfamily A strain that is heterologous toa N. meningitidis strain expressing A05. In one embodiment, the immuneresponse is against N. meningitidis serogroup B A22 strain. In oneembodiment, the immune response is against N. meningitidis serogroup BA56 strain. In one embodiment, the immune response is against N.meningitidis serogroup B A06 strain. In one embodiment, the immuneresponse is against N. meningitidis serogroup B A15 strain. In oneembodiment, the immune response is against N. meningitidis serogroup BA29 strain. In one embodiment, the immune response is against N.meningitidis serogroup B A62 strain. In one embodiment, the immuneresponse is bactericidal against a N. meningitidis serogroup B subfamilyA strain that is heterologous to N. meningitidis strain M98250771.

In one embodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily A strain that expresses a factor Hbinding protein including an amino acid sequence that has at least 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identity to the first polypeptide. Inanother embodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily A strain that expresses a factor Hbinding protein including an amino acid sequence that has at least 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identity to a factor H binding proteinexpressed by N. meningitidis strain M98250771. In a preferredembodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily A strain that expresses a factor Hbinding protein including an amino acid sequence that has at least 80%,more preferably at least 84%, identity to a factor H binding proteinexpressed by N. meningitidis strain M98250771.

In another embodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily A strain that expresses a factor Hbinding protein including an amino acid sequence that has at most 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identity to the first polypeptide. Inanother embodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily A strain that expresses a factor Hbinding protein including an amino acid sequence that has at most 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identity to a factor H binding proteinexpressed by N. meningitidis strain M98250771. In a preferredembodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily A strain that expresses a factor Hbinding protein including an amino acid sequence that has at most 85%,more preferably at most 99%, identity to a factor H binding proteinexpressed by N. meningitidis strain M98250771. Any minimum value may becombined with any maximum value described herein to define a range.

In one embodiment, the immune response elicited by the composition isbactericidal not only against a N. meningitidis serogroup B fHPBsubfamily A strain but also a N. meningitidis strain expressing an fHBPsubfamily A polypeptide, wherein the serogroup is not serogroup B. Forexample, in one preferred embodiment, the immune response elicited bythe composition is bactericidal against a N. meningitidis serogroup Bsubfamily A strain and against a N. meningitidis serogroup C strain thatexpresses an fHBP subfamily A polypeptide heterologous to fHBP A05. Forexample, in one embodiment, the immune response is against a N.meningitidis serogroup C strain expressing fHBP A10. In anotherembodiment, the immune response is against a N. meningitidis serogroup Wstrain expressing fHBP A19. In one embodiment, the immune response isbactericidal against a N. meningitidis strain that expresses an fHBPsubfamily A polypeptide, wherein the strain is heterologous to N.meningitidis strain M98250771.

Subfamily B Strains

In one embodiment, the hSBA strain is an LP2086 (fHBP) subfamily Bstrain. In one embodiment, the hSBA strain is an LP2086 (fHBP) subfamilyB strain that expresses a lipoprotein 2086 variant that is heterologousto a N. meningitidis strain expressing B01. For example, in oneembodiment, the hSBA strain is an LP2086 (fHBP) subfamily B strain thatexpresses a lipoprotein 2086 variant that is heterologous to strainCDC1127. In a preferred embodiment, the hSBA strain is an LP2086 (fHBP)subfamily B strain that expresses a lipoprotein 2086 variant that isheterologous to strain CDC1573.

In one embodiment, the immune response is bactericidal against a N.meningitidis serogroup B fHPB subfamily B strain that is heterologous toa N. meningitidis strain expressing B01. In one embodiment, the immuneresponse is against N. meningitidis serogroup B B24 strain. In oneembodiment, the immune response is against N. meningitidis serogroup BB44 strain. In one embodiment, the immune response is against N.meningitidis serogroup B B16 strain. In one embodiment, the immuneresponse is against N. meningitidis serogroup B B03 strain. In oneembodiment, the immune response is against N. meningitidis serogroup BB09 strain. In one embodiment, the immune response is against N.meningitidis serogroup B B15 strain. In one embodiment, the immuneresponse is against N. meningitidis serogroup B B153 strain. In oneembodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily B strain that is heterologous to N.meningitidis strain CDC1573.

In one embodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily B strain that expresses a factor Hbinding protein including an amino acid sequence that has at least 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identity to the second polypeptide. Inanother embodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily B strain that expresses a factor Hbinding protein including an amino acid sequence that has at least 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identity to a factor H binding proteinexpressed by N. meningitidis strain CDC1573. In a preferred embodiment,the immune response is bactericidal against a N. meningitidis serogroupB subfamily B strain that expresses a factor H binding protein includingan amino acid sequence that has at least 80% identity, more preferablyat least 87% identity, to a factor H binding protein expressed by N.meningitidis strain CDC1573. In another preferred embodiment, the immuneresponse is bactericidal against a N. meningitidis serogroup B subfamilyB strain that expresses a factor H binding protein including an aminoacid sequence that has 100% identity to a factor H binding proteinexpressed by N. meningitidis strain CDC1573.

In another embodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily B strain that expresses a factor Hbinding protein including an amino acid sequence that has at most 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identity to the second polypeptide. Inanother embodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily B strain that expresses a factor Hbinding protein including an amino acid sequence that has at most 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identity to a factor H binding proteinexpressed by N. meningitidis strain CDC1573. In a preferred embodiment,the immune response is bactericidal against a N. meningitidis serogroupB subfamily B strain that expresses a factor H binding protein includingan amino acid sequence that has at most 88% identity, more preferably atleast 99% identity, to a factor H binding protein expressed by N.meningitidis strain CDC1573. Any minimum value may be combined with anymaximum value described herein to define a range.

In one embodiment, the hSBA strain is an LP2086 (fHBP) B24 strain. Inanother embodiment, the hSBA strains is an LP2086 (fHBP) B44 strain. Ina further embodiment, the hSBA strains includes LP2086 (fHBP) B24 andLP2086 (fHBP) B44 strains. In one embodiment, the hSBA strains includesLP2086 (fHBP) A22, LP2086 (fHBP) A56, LP2086 (fHBP) B24, and LP2086(fHBP) B44 strains. In one embodiment, the hSBA strain includes B15. Inone embodiment, the hSBA strain includes B153. In another embodiment,the hSBA strain is an LP2086 B16 strain. In one embodiment, the hSBAstrain is an LP2086 B03 strain. In one embodiment, the hSBA strain is anLP2086 B09 strain. In a further embodiment, the hSBA strains includeB24, B16, B44, B03, and B09, or any combination thereof. In anotherembodiment, the hSBA strains include B24, B16, B44, A22, B03, B09, A12,A19, A05, and A07, or any combination thereof. In another embodiment,the hSBA strains include A06, A07, A12, A15, A19, A29, B03, B09, B15,and B16, or any combination thereof.

In one embodiment, the method induces an immune response against a N.meningitidis serogroup B fHPB subfamily A strain and against a N.meningitidis serogroup B fHPB subfamily B strain. Preferably, the immuneresponse is bactericidal against a N. meningitidis serogroup B fHPBsubfamily A strain and against a N. meningitidis serogroup B fHPBsubfamily B strain.

In one embodiment, the immune response elicited by the composition isbactericidal not only against a N. meningitidis serogroup B fHPBsubfamily B strain but also a N. meningitidis strain expressing an fHBPsubfamily B polypeptide, wherein the serogroup is not serogroup B. Forexample, in one preferred embodiment, the immune response elicited bythe composition is bactericidal against a N. meningitidis serogroup Bsubfamily B strain and against a N. meningitidis serogroup Y strain thatexpresses an fHBP subfamily B polypeptide heterologous to fHBP B01. Forexample, in one embodiment, the immune response is against a N.meningitidis serogroup A strain expressing fHBP B16. In anotherembodiment, the immune response is against a N. meningitidis serogroup Ystrain expressing fHBP B47. In another embodiment, the immune responseis against a N. meningitidis serogroup X strain expressing fHBP B49. Inone embodiment, the immune response is bactericidal against a N.meningitidis strain that expresses an fHBP subfamily B polypeptide,wherein the strain is heterologous to N. meningitidis serogroup B strainCDC1573.

Titers

In one embodiment, the composition induces an increase in bactericidaltiter in the human, as compared to the bactericidal titer in the humanprior to administration of a dose of the composition, when measuredunder identical conditions in an hSBA. In one embodiment, the increasein bactericidal titer is compared to the bactericidal titer in the humanbefore administration of the first dose of the composition, as comparedto the bactericidal titer in the human prior to administration of thefirst dose of the composition, when measured under identical conditionsin an hSBA. In one embodiment, the increase in titer is observed after asecond dose of the composition, as compared to the bactericidal titer inthe human prior to administration of the second dose of the composition,when measured under identical conditions in an hSBA. In anotherembodiment, the increase in bactericidal titer is observed after a thirddose of the composition, as compared to the bactericidal titer in thehuman prior to administration of the third dose of the composition, whenmeasured under identical conditions in an hSBA.

In one embodiment, the composition induces a bactericidal titer in thehuman after administration of a dose, wherein the bactericidal titer isat least greater than 1-fold higher than the bactericidal titer in thehuman prior to administration of the dose, when measured under identicalconditions in an hSBA. For example, the bactericidal titer may be atleast 1.01-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold,14-fold, 15-fold, or 16-fold higher in the human after receiving a doseof the composition, as compared to the bactericidal titer in the humanprior to administration of the dose, when measured under identicalconditions in an hSBA.

In one embodiment, a “responder” refers to a human, wherein thecomposition induces a bactericidal titer in the human afteradministration of a dose, wherein the bactericidal titer is at leastgreater than 1-fold higher than the bactericidal titer in the humanprior to administration of the dose. In a preferred embodiment, theresponder achieves at least a ≥4-fold rise in hSBA titer, as compared toa bactericidal titer in the human prior to administration of the dose.Such a responder may be referred to as having a protective titer. Insome embodiments, a protective titer is one that is greater than 1:4.

In one embodiment, the hSBA titer is the reciprocal of the highestdilution of a serum sample that produces a measurable effect. Forexample, in one embodiment, the hSBA titer is the reciprocal of thehighest 2-fold dilution of a test serum that results in at least a 50%reduction of MnB bacteria (50% bacterial survival) compared to the T30CFU value (i.e., the number of bacteria surviving after incubation inassay wells containing all assay components except test serum; 100%bacterial survival).

In one embodiment, the composition induces a bactericidal titer in thehuman after receiving the first dose that is at least 2-fold higher thanthe bactericidal titer in the human prior to receiving the first dose(e.g., higher than the bactericidal titer in the human in the absence ofthe first dose), when measured under identical conditions in the hSBA.In one embodiment, the composition induces a bactericidal titer in thehuman that is at least 4-fold higher than the bactericidal titer in thehuman prior to receiving the first dose, when measured under identicalconditions in a human serum bactericidal assay that utilizes humancomplement (hSBA). In one embodiment, the composition induces abactericidal titer in the human that is at least 8-fold higher than thebactericidal titer in the human prior to receiving the first dose, whenmeasured under identical conditions in a human serum bactericidal assaythat utilizes human complement (hSBA).

In a preferred embodiment, the human serum complement is derived from ahuman having low intrinsic bactericidal activity for a given hSBA teststrain. Low intrinsic bactericidal activity refers to, for example, abactericidal titer that is at least less than a 1:4 dilution against thegiven hSBA test strain. In one embodiment, the human complement isderived from a human having an hSBA titer that is at least less than1:4, such as a 1:2 dilution, against the given hSBA test strain, whereinthe composition was not administered to the human.

A human may exhibit an hSBA titer of less than 1:4 prior toadministration of a composition, such as the bivalent rLP2086composition, or a human may exhibit an hSBA titer of ≥1:4 prior toadministration of the composition. Accordingly, in preferred embodimentsand examples, administration of at least one dose of the composition tothe human results in an hSBA titer that is at least 4-fold greater thanthe titer in the human prior to the administration. In some embodiments,administration of at least one dose of the composition to the humanresults in an hSBA titer that is at least greater than 1:4, such as, forexample, an hSBA titer of ≥1:8, an hSBA titer of ≥1:16, and an hSBAtiter of ≥1:32. The respective Examples described herein includeassessments of the proportion of human subjects having an hSBA titer≥1:8 and/or ≥1:16, wherein the bivalent rLP2086 composition wasadministered to the human. In some embodiments, a 4-fold rise in titerin the human after administration of the composition as compared tobefore administration of the composition show that protection isassociated with the composition. In some embodiments, such preferredassessments of hSBA titers greater than 1:4 show that the protection,i.e., the bactericidal immune response induced in the human, isassociated with the composition.

In one embodiment, the human has an hSBA titer equal to or greater thanthe hSBA's lower limit of quantitation (LLOQ) after administration ofthe first dose of the composition. In another embodiment, the human hasan hSBA titer equal to or greater than the hSBA's LLOQ afteradministration of the second dose of the composition. In anotherembodiment, the human has an hSBA titer equal to or greater than thehSBA's LLOQ after administration of the third dose of the composition.

Methods and Administration

In one aspect, the invention relates to a method of inducing an immuneresponse against N. meningitidis in a human. In another aspect, theinvention relates to a method of vaccinating a human. In one embodiment,the method includes administering to the human at least one dose of thecomposition described above. In a preferred embodiment, the methodincludes administering to the human at most one dose of the compositiondescribed above. In another embodiment, the method includesadministering to the human at least a first dose and a second dose ofthe composition described above.

In one embodiment, the second dose is administered at least 20, 30, 50,60, 100, 120, 160, 170, or 180 days after the first dose, and at most250, 210, 200, or 190 days after the first dose. Any minimum value maybe combined with any maximum value described herein to define a range.

In another embodiment, the second dose is administered about 30 daysafter the first dose. In another embodiment, the second dose isadministered about 60 days after the first dose, such as, for example,in a 0, 2 month immunization schedule. In another embodiment, the seconddose is administered about 180 days after the first dose, such as, forexample, in a 0, 6 month immunization schedule. In yet anotherembodiment, the second dose is administered about 120 days after thefirst dose, such as, for example, in a 2, 6 month immunization schedule.

In one embodiment, the method includes administering to the human twodoses of the composition and at most two doses. In one embodiment, thetwo doses are administered within a period of about 6 months after thefirst dose. In one embodiment, the method does not include furtheradministration of a booster to the human. A “booster” as used hereinrefers to an additional administration of the composition to the human.Administering to the human at most two doses of the composition may beadvantageous. Such advantages include, for example, facilitating a humanto comply with a complete administration schedule and facilitatingcost-effectiveness of the schedule.

In one embodiment, the first dose and the second dose are administeredto the human over a period of about 25, 30, 40, 50, 60, 70, 80, 90, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 days, and most 400,390, 380, 370, 365, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260,250, 240, 230, 220, 210, or 200 days after the first dose. Any minimumvalue may be combined with any maximum value described herein to definea range. Preferably, the first and second doses will be administered atleast 4 weeks apart e.g. ≥8 weeks apart, ≥2 months apart, ≥3 monthsapart, ≥6 months apart, etc.

In one embodiment, the first dose and the second dose are administeredto the human over a period of about 30 days. In another embodiment, thefirst dose and the second dose are administered to the human over aperiod of about 60 days. In another embodiment, the first dose and thesecond dose are administered to the human over a period of about 180days.

Conveniently, the first dose can be administered at substantially thesame time as (e.g. during the same medical consultation or visit to ahealthcare professional or within 24 hours of the first dose of themeningococcal vaccine) another vaccine e.g. at substantially the sametime as a hepatitis B virus vaccine, a diphtheria vaccine, a tetanusvaccine, a pertussis vaccine (either cellular or, preferably,acellular), a Haemophilus influenzae type b vaccine, a Streptococcuspneumoniae vaccine, and/or a polio vaccine (preferably in inactivatedpoliovirus vaccine). Each of these optionally co-administered vaccinesmay be a monovalent vaccine or may be part of a combination vaccine(e.g. as part of a DTP vaccine).

Conveniently, the second dose can be administered at substantially thesame time as (e.g. during the same medical consultation or visit to ahealthcare professional or within 24 hours of the second dose of themeningococcal vaccine) another vaccine e.g. at substantially the sametime as a hepatitis B virus vaccine, a diphtheria vaccine, a tetanusvaccine, a pertussis vaccine (either cellular or acellular), aHaemophilus influenzae type b vaccine, a Streptococcus pneumoniaevaccine, a polio vaccine (preferably in inactivated poliovirus vaccine),an influenza vaccine, a chickenpox vaccine, a measles vaccine, a mumpsvaccine, and/or a rubella vaccine. Each of these optionallyco-administered vaccines may be a monovalent vaccine or may be part of acombination vaccine (e.g. as part of an MMR vaccine).

Conveniently, the third dose can be administered at substantially thesame time as (e.g. during the same medical consultation or visit to ahealthcare professional or within 24 hours of the third dose of themeningococcal vaccine) another vaccine e.g. at substantially the sametime as a hepatitis B virus vaccine, a diphtheria vaccine, a tetanusvaccine, a pertussis vaccine (either cellular or acellular), aHaemophilus influenzae type b vaccine, a Streptococcus pneumoniaevaccine, a polio vaccine (preferably in inactivated poliovirus vaccine),an influenza vaccine, a chickenpox vaccine, a measles vaccine, a mumpsvaccine, and/or a rubella vaccine. Each of these optionallyco-administered vaccines may be a monovalent vaccine or may be part of acombination vaccine (e.g. as part of an MMR vaccine).

Three Doses

In one embodiment, a three-dose schedule of the composition induces abactericidal titer against multiple strains expressing LP2086 (fHBP)heterologous to the first and/or second polypeptide in a greaterpercentage of humans than a two-dose schedule.

In one embodiment, the method includes administering to the human threedoses of the composition. In another embodiment, the method includesadministering at most three doses of the composition. In one embodiment,the three doses are administered within a period of about 6 months afterthe first dose. In one embodiment, the method includes an administrationof a booster dose to the human after the third dose. In anotherembodiment, the method does not include administration of a booster doseto the human after the third dose. In another embodiment, the methoddoes not further include administering a fourth or booster dose of thecomposition to the human. In a further embodiment, at most three doseswithin a period of about 6 months are administered to the human.

In an exemplary embodiment, the second dose is administered about 30days after the first dose, and the third dose is administered about 150days after the second dose, such as, for example, in a 0, 1, 6 monthimmunization schedule. In another exemplary embodiment, the second doseis administered about 60 days after the first dose, and the third doseis administered about 120 days after the second dose, such as, forexample, in a 0, 2, 6 month immunization schedule.

In one embodiment, the first dose, second dose, and third dose areadministered to the human over a period of about 150, 160, 170, or 180days, and at most 240, 210 200, or 190 days. Any minimum value may becombined with any maximum value described herein to define a range.Preferably, the first dose, second dose, and third dose is administeredto the human over a period of about 180 days or 6 months. For example,the second dose may be administered to the human about 60 days after thefirst dose, and the third dose may be administered to the human about120 days after the second dose. Accordingly, an exemplary schedule ofadministration includes administering a dose to the human at aboutmonths 0, 2, and 6.

As described above, multiple doses of the immunogenic composition may beadministered to the human, and the number of days between each dose mayvary. An advantage of the method includes, for example, flexibility fora human to comply with the administration schedules.

In one embodiment, the method includes administering to the human atmost three doses of the identical immunogenic composition. For example,in a preferred embodiment, the method does not include administering tothe human a first dose of a first composition, administering to thehuman a second dose of a second composition, and administering to thehuman a third dose of a third composition, wherein the first, second,and third compositions are not identical. In another embodiment, themethod includes administering to the human at most four doses of theidentical immunogenic composition.

EXAMPLES

The following Examples illustrate embodiments of the invention. Unlessnoted otherwise herein, reference is made in the following Examples to aMnB bivalent rLP2086 composition, at the 120-μg bivalent rLP2086 doselevel, which is a preferred exemplary embodiment of a compositionincluding: 60 μg of a first lipidated polypeptide including the aminoacid sequence set forth in SEQ ID NO: 1 per 0.5 mL dose, 60 μg of asecond lipidated polypeptide including the amino acid sequence set forthin SEQ ID NO: 2 per 0.5 mL dose, 2.8 molar ratio polysorbate-80 to thefirst polypeptide, 2.8 molar ratio polysorbate-80 to the secondpolypeptide, 0.5 mg Al³⁺/ml of the composition, 10 mM histidine, and 150mM sodium chloride.

More specifically, the investigational bivalent recombinant rLP2086vaccine at the 120-μg bivalent rLP2086 dose level includes (a) 60 μg ofa first lipidated polypeptide including the amino acid sequence setforth in SEQ ID NO: 1; (b) 60 μg of a second lipidated polypeptideincluding the amino acid sequence set forth in SEQ ID NO: 2; (c) 18 μgpolysorbate-80; (d) 250 μg aluminum; (e) 780 μg histidine, and (f) 4380μg sodium chloride. Each dose is 0.5 mL.

Unless noted otherwise herein, reference is made in the followingExamples to a MenACWY-TT composition, which is a preferred exemplaryembodiment of a tetravalent meningococcal polysaccharide conjugatedcomposition that includes Neisseria meningitidis capsularpolysaccharides A, C, W-135 and Y each coupled to tetanus toxoid as acarrier protein. The Neisseria meningitidis serogroups A and Cpolysaccharides are conjugated with an adipic dihydrazide (AH) spacerand indirectly conjugated to the tetanus toxoid whereas the W-135 and Ypolysaccharides are conjugated directly to tetanus toxoid. Thecomposition does not contain any preservatives or adjuvants.

More specifically, the lyophilized MenACWY-TT composition described inthe examples below includes 5 micrograms of Neisseria meningitidisserogroup A polysaccharide conjugated to tetanus toxoid carrier protein;5 micrograms of Neisseria meningitidis serogroup C polysaccharideconjugated to tetanus toxoid carrier protein; 5 micrograms of Neisseriameningitidis serogroup W-135 polysaccharide conjugated to tetanus toxoidcarrier protein; 5 micrograms of Neisseria meningitidis serogroup Ypolysaccharide conjugated to tetanus toxoid carrier protein; 28 mgsucrose; 97 μg trometamol, per dose (0.5 mL).

Example 1: The MenABCWY Composition

The final MenABCWY composition is prepared by reconstituting thelyophilized MenACWY-TT Drug Product (described in Example 2 below) vialwith 0.67 mL of MnB Bivalent rLP2086 Drug Product (described in Example3 below) in order to withdraw 0.5 mL dose of MenABCWY vaccine forintramuscular injection. All components used in the preparation of theMenABCWY vaccine and their functions are provided in Table 1 below.

TABLE 1 Composition of MenABCWY vaccine Ingredients Amount/dose MnBrLP2086 subfamily A 60 mcg (SEQ ID NO: 1) MnB rLP2086 subfamily B 60 mcg(SEQ ID NO: 2) MenA_(AH)-TT conjugate 5 mcg MenA (mean TT/polysaccharideratio: ~3) ~7.5 mcg TT MenC_(AH)-TT conjugate 5 mcg MenC (meanTT/polysaccharide ratio: ~3) ~7.5 mcg TT MenW-TT conjugate 5 mcg MenW(mean TT/polysaccharide ratio: ~1.5) ~3.75 mcg TT MenY-TT conjugate 5mcg MenY (mean TT/polysaccharide ratio: ~1.3) ~3.25 mcg TT Tris-HCl 97mcg Sodium Chloride^(a) 4.69-4.71 mg Sucrose 28 mg L-Histidine 0.78 mgPolysorbate 80 (PS80) 0.02 mg Aluminum phosphate 0.25 mg aluminum Waterfor injection qs to 0.5 mL ^(a)Sodium chloride concentration in MenABCWYVaccine may vary between 160.5-161.1 mM based on the composition of theclinical and commercial NIMENRIX Drug Product (DP) lots.

Example 2: Description and Composition of the MnB Bivalent Rlp2086 DrugProduct

MnB bivalent rLP2086 drug product is a sterile liquid formulationcomposed of rLP2086 subfamily A and B proteins formulated at 120μg/mL/subfamily in 10 mM histidine buffer, 150 mM sodium chloride (NaCl)at pH 6.0 with 0.5 mg/mL aluminum as aluminum phosphate (AlPO4).Polysorbate 80 (PS-80) is added to drug substance to obtain the targetPS-80 to protein molar ratio. Therefore, PS-80 is not added during thedrug product formulation but is present in the final drug product at thesame ratio. The drug product is filled into 1 mL syringes. A single doseof vaccine is 0.5 mL with no preservatives.

TABLE 2 Composition of MnB Bivalent rLP2086 Drug Product IngredientsQuantity/dose MnB rLP2086 subfamily A (SEQ ID NO: 1) 120 μg/mL MnBrLP2086 subfamily B (SEQ ID NO: 2) 120 μg/mL Sodium chloride 150 mML-Histidine 10 mM Aluminum phosphate 0.50 mg Aluminum phosphate/mL Waterfor injection qs to 1 mL ^(a)Polysorbate 80 (PS-80) is part of drugsubstance. PS-80 functions as a surfactant in the drug product.^(b)Equivalent to 0.25 mg aluminum per dose

The Effect of Polysorbate 80 Concentration

Polysorbate 80 (PS-80) is a non-ionic surfactant. It is used tostabilize and solubilize MnB rLP2086 subfamily A and B proteins in theformulation by preventing aggregation and adsorption that may be causedby temperature, filter, tubing, container/closure contact and processmixing. Stability studies using an in vitro monoclonal antibody basedpotency assay demonstrated instability of the subfamily B protein athigher molar ratios of PS-80 to MnB rLP2086 protein in the finalformulation. Experiments with varying molar ratios of PS-80 to proteinhave demonstrated that the optimal molar ratio of PS-80 to MnB rLP2086protein is approximately between 1.4 to 4.2 to retain potency.

Example 3: Description and Composition of the MenACWY-TT Composition

MenACWY-TT drug product is composed of the purified polysaccharides ofNeisseria meningitidis serogroups A, C, W and Y, each conjugated toTetanus Toxoid (TT) at ratios to polysaccharide of ˜3, ˜3, ˜1.5 and˜1.3, respectively.

The MenACWY-TT drug product is presented as a lyophilized powder,supplied in a 3 mL glass vial with bromobutyl rubber closures suitablefor lyophilization and aluminum flip-off caps. All components used inthe manufacture of the MenACWY-TT Drug product and their functions areprovided in Table 3.

TABLE 3 Composition of MenACWY-TT Drug product Ingredients Quantity/doseMenA_(AH)-TT conjugate 5 mcg MenA (mean TT/polysaccharide ratio: ~3) ~15mcg TT MenC_(AH)-TT conjugate 5 mcg MenC (mean TT/polysaccharide ratio:~3) ~15 mcg TT MenW-TT conjugate 5 mcg MenW (mean TT/polysaccharideratio: ~1.5) ~7.5 mcg TT MenY-TT conjugate 5 mcg MenY (meanTT/polysaccharide ratio: ~1.3) ~6.5 mcg TT Tris-HCl 97 mcg Sucrose 28 mgSodium Chloride^(a) 306.0-325.0 mg ^(a)Lyophilized cake also containssodium chloride resulting from the salt present in each of the bulkpurified TT conjugates. Sodium chloride concentration varies between10.5-11.1 mM based on the composition of the clinical and commerciallots.

Example 4: Preparation of the MenABCWY Composition

The final MenABCWY composition is prepared in the clinic byreconstituting the lyophilized MenACWY-TT drug product vial with 0.67 mLof MnB Bivalent rLP2086. The resulting MenABCWY composition (a vaccineliquid drug product) contains rLP2086 subfamily A and B proteins at 120mcg/mL/subfamily, purified polysaccharides of Neisseria meningitis typesA, C, W and Y at concentration of 10 mcg/mL/type conjugated to TetanusToxoid at ratios of ˜3, ˜3, ˜1.5, and ˜3 respectively in 10 mM histidineand 1.6 mM tris buffer containing 160.5-161.1 mM sodium chloride, 0.5mg/mL aluminum as aluminum phosphate (AIP04), 0.035 mg/mL polysorbate 80and 56 mg/mL sucrose at pH of 6.05 for intramuscular injection.

The MenABCWY vaccine is prepared by mixing of two drug products,MenACWY-TT and MnB Bivalent rLP2086. Buffering components and excipientswere chosen based on the individual development of each component andare shown to provide the necessary stability profile for extended shelflife.

Dosage verification studies were performed to demonstrate thatMenACWY-TT drug product and MnB Bivalent rLP2086 drug product arecompatible when mixed together for administration of MenABCWY vaccineand that all drug product and dosing solutions are compatible with theadministration components and that dosing solutions are stable in theadministration components for a period of time adequate to perform thedose preparation and administration operations. The stability ofMenABCWY vaccine prepared by reconstitution of MenACWY-TT drug productwith 0.67 mL of MnB Bivalent rLP2086 drug product over the hold time atambient temperature and light conditions was confirmed in reconstitutedvials and in dosing syringes.

Samples representing the dosing solutions of MenABCWY vaccine weretested using stability indicating methods such as RP-HPLC for antigenbinding and purity, bioplex activity assay, ELISA, and ICP-MS withpredefined acceptance criteria. The results of this study showacceptable stability of MenABCWY vaccine for 24 hours at roomtemperature and light conditions. These data are shown and described inExamples 5-15 below.

Example 5: Evaluation of the MenABCWY Vaccine

A study was carried out to assess whether there is acceptable physicalcompatibility and short-term stability when a lyophilized MenACWY-TTcomposition is reconstituted with the MnB bivalent rLP2086 composition.The lyophilized MenACWY-TT composition and the liquid MnB bivalentrLP2086 composition were combined and stored for up to 24 hours in anuncontrolled room temperature environment to approximate real lifeconditions. It was demonstrated that lyophilized MenACWY-TT compositioncould be reconstituted with the liquid MnB bivalent rLP2086 compositionwith gentle hand mixing and the combined pH and osmolality were withintypical range for an injectable. All key attributes for the conjugatesand proteins were similar to those of a control for up to 24 hours inthe uncontrolled room temperature environment.

The physical compatibility was evaluated through assessing pH,appearance, ease of reconstitution, and osmolality of the combined drugproduct. The stability of the antigens was evaluated through assessingconcentration, purity and the in-vitro relative antigenicity (IVRA) ofthe rLP2086 subfamily A and subfamily B proteins as well as theconcentrations of the conjugated Meningococcal A, C, Y, and W-135polysaccharides by ELISA.

Example 5 through Example 15 demonstrate that the combination of thelyophilized MenACWY-TT composition and the liquid MnB bivalent rLP2086composition, i.e., the MenABCWY composition, was found to be compatibleand stable for at least 24 hours at room temperature.

ELISAs for Determining Mening A, C, Y, and W-135 PolysaccharideConcentrations in the MenABCWY Composition—Development of the Mening A,C, Y and W-135 ELISA & Screening of the pAb for Detection

Six antibodies were selected for screening for use in the ELISA assays.Each of four groups of ten rabbits was immunized with either Men A, C, Yor W-135 polysaccharide TT conjugates with rabbits subsequentlyexsanguinated after antibody development. Each rabbit was individuallyscreened using Men A, C, Y or W-135 polysaccharides conjugated to acarrier protein CRM₁₉₇ for binding and specificity. Rabbit sera wasscreened for a positive binding signal, which is equivalent to anabsorbance reading greater than three-fold above the backgroundabsorbance. Additionally, rabbit sera was screened for low non-specificbinding, which was any absorbance readings for sera combinations withoutantigen, secondary or detection above the absorbance reading for theblank, as well as low cross-reactivity, which was any absorbancereadings for heterologous serotypes that was above the backgroundabsorbance. Rabbits that met the screening criteria were pooled. Thestandard curve range was established using CRM conjugates and confirmedwith reconstituted the lyophilized MenACWY-TT composition. The standardcurve range was established using CRM conjugates and confirmed withreconstituted lyophilized MenACWY-TT composition.

The feasibility of quantitating the A, C, Y and W conjugates in thecombined drug product (MenABCWY composition) was established. It wasdetermined that the MnB Bivalent rLP2086 composition alone was notdetected in the assay. Additionally, when aluminum phosphate in theMenABCWY composition samples was solubilized, full recovery ofconjugates was obtained. Therefore, it was determined that the MnBbivalent rLP2086 composition does not interfere with the quantitation ofthe MenACWY-TT conjugates by ELISA.

Example 6: Evaluation of Suitability of Methods to Assess the MnBBivalent rLP2086 Composition in the Presence of the MenACWY-TTComposition

IEX-HPLC was evaluated for its suitability to determine the strength ofthe MnB bivalent rLP2086 composition subfamily A and B proteins in thepresence of the MenACWY-TT composition. The total protein and boundprotein results for the MnB bivalent rLP2086 composition in the presenceof the MenACWY-TT composition and in the absence of the MenACWY-TTcomposition were accessed. The overlaid chromatograms are shown in FIG.1.

TABLE 4 Sample Subfamily Bound Protein, % the MnB bivalent Protein A(SEQ ID 107 rLP2086 NO: 1) composition Protein B (SEQ ID 104 no theMenACWY- NO: 2) TT composition the MnB bivalent Protein A (SEQ ID 108rLP2086 NO: 1) composition Protein B (SEQ ID 103 with the NO: 2)MenACWY-TT composition

Example 7: Evaluation of the MnB Bivalent rLP2086 Composition Purity andPeak Ratio in the Presence of the MenACWY-TT Composition

RP-HPLC was evaluated for its suitability to determine the purity of theMnB bivalent rLP2086 composition in the presence of the MenACWY-TTcomposition. The purity results for the MnB bivalent rLP2086 compositionin the presence of the MenACWY-TT composition and in the absence of theMenACWY-TT composition were compared. The overlaid chromatograms areshown in FIG. 2. An example of the integration of the impurity peak isshown as an insert in FIG. 2. The evaluation results show that thepresence of the MenACWY-TT composition does not interfere withevaluation of the MnB bivalent rLP2086 composition purity using theRP-HPLC method.

Example 8: Evaluation of the MnB Bivalent rLP2086 Composition IVRA inthe Presence of the MenACWY-TT Composition

The IVRA method was evaluated for its suitability for determination ofin-vitro relative antigenicity of the MnB bivalent rLP2086 compositionSubfamily A (SEQ ID NO: 1) and Subfamily B (SEQ ID NO: 2) proteins inthe presence of the MenACWY-TT composition.

The IVRA results for the MnB bivalent rLP2086 composition Subfamily Aand Subfamily B proteins in the presence and in the absence of theMenACWY-TT composition were compared. The feasibility evaluation resultsshow that, within the assay variability the results are comparable andthat the presence of the MenACWY-TT composition does not interfere withdetermination of in-vitro relative antigenicity.

Example 9: Reconstitution of the MenACWY-TT Composition Vials with theMnB Bivalent rLP2086 Composition

The MenACWY-TT composition and the MnB bivalent rLP2086 composition drugproducts were performed using the MenACWY-TT composition vialsreconstituted with the MnB bivalent rLP2086 composition drug product.The MenACWY-TT composition vials reconstituted with either saline or theMenACWY-TT composition matrix placebo were used as controls depending onthe method.

TABLE 5 Product Component Composition The MenACWY- Neisseriameningitidis TT composition Group A polysaccharide 10 μg/mL Group Cpolysaccharide 10 μg/mL Group W-135 polysaccharide 10 μg/mL Group Ypolysaccharide 10 μg/mL Tetanus toxoid carrier protein 88 μg/mL Sucrose164 mM Trometanol 1.6 mM the MnB bivalent Sub-family A rLP2086 protein120 μg/mL rLP2086 (SEQ ID NO: 1) composition Sub-family B rLP2086protein 120 μ/mL (SEQ ID NO: 2) AlPO4 0.5 mg/mL Histidine 10 mM NaCl 150mM pH 6.0 the MnB bivalent AlPO4 0.5 mg/mL rLP2086 Histidine 10 mMcomposition NaCl 150 mM Placebo pH 6.01 Bulk MnB Sub-family A rLP2086protein 120 μg/mL bivalent rLP2086 (SEQ ID NO: 1) composition DPSub-family B rLP2086 protein 120 μg/mL (SEQ ID NO: 2) AlPO4 0.5 mg/mLHistidine 10 mM NaCl 150 mM pH 6.0

Determination of Saline Reconstitution Volume for the MenACWY-TTComposition

The NIMENRIX® commercial product package contains both a vial containingthe lyophilized MenACWY-TT composition and a syringe containing 0.9%saline used for reconstitution. In order to reproduce the finalNIMENRIX® concentration in the commercial vaccine upon reconstitutionwith the MnB bivalent rLP2086 composition, the amount of salinedispensed using the syringe from the commercial product had to bedetermined. This same volume of the MnB bivalent rLP2086 compositionwould then be used for all reconstitution studies.

Reconstitution of the MenACWY-TT Composition Vials with the MnB BivalentrLP2086 Composition

The MnB bivalent rLP2086 composition was pooled in a 10 mL glass vial.Approximately 800 μL of the solution was withdrawn into a 1 mL syringe.The adjusted contents of the syringe were injected into a vialcontaining the MenACWY-TT composition. The vial was swirled to dissolvethe contents.

The pH and appearance were determined on duplicate samples on theMenABCWY composition. Osmolality was measured in triplicate on theMenACWY-TT composition reconstituted with saline and on the MenACWY-TTcomposition reconstituted with the MnB bivalent rLP2086 composition.

Example 10: SEC-MALLS to Evaluate Mening A, C, Y, and W-135Polysaccharide Stability in DP Matrix

Mening A, C, Y, and W-135 Polysaccharides were used as surrogates toassess if any instability of the conjugated Meningococcal A, C, Y, andW-135 polysaccharides in the combined drug product (the MenABCWYcomposition) could be expected.

Treatment of Mening a, C, Y, and W-135 Polysaccharides

Reagent Preparation (“Full MenABCWY Composition Buffer Matrix”)

2.24 μg of sucrose and 7.8 mg of Tris (Tromethamine) was added to 20 mlof 2×MnB bivalent rLP2086 composition buffer matrix with MnB rLP2086proteins (Histidine 20 mM pH 6.0, NaCl 300 mM, PS 80 0.07 mg/ml, AlPO4 1mg/ml (8 mM), rLP2086 subfamily A (SEQ ID NO: 1) and subfamily B (SEQ IDNO: 2) proteins 240 μg/mL each).

Sample Preparation

Each Mening Polysaccharide was diluted 1:1 with Full MenABCWYcomposition Buffer Matrix and incubated for 0, 6 and 24 hours at 5° C.,25° C. and 37° C. After incubation the sample suspension was spun for 1minute at 14,000 r.p.m. The supernatant was analyzed by SEC-MALLS.

Example 11: Stability of the MenABCWY Composition—Evaluation of pH,Appearance, and Osmolality of the Combined MnB Bivalent rLP2086 andMenACWY-TT Compositions

The pH and appearance of the combined MnB bivalent rLP2086 compositionand the MenACWY-TT composition, i.e., the MenABCWY composition, wereevaluated immediately after reconstitution and again after 24 hours.

All results were as expected (Table 6).

TABLE 6 Appearance and pH of the MenABCWY composition Time Point, Sample# Sample hours Appearance pH 1 MenABCWY 0 Homogeneous 5.8 composition,white Rep1 suspension 2 MenABCWY 0 Homogeneous 5.8 composition, whiteRep2 suspension 3 MenABCWY 24 Homogeneous 5.8 composition, white Rep 1suspension 4 MenABCWY 24 Homogeneous 5.8 composition, white Rep 2suspension 5 MenACWY-TT 0 Clear, 6.3 composition Colorless w/Saline 6MenACWY-TT 24 Clear, 6.4 composition Colorless w/SalineThe average osmolality of the MenACWY-TT composition reconstituted withthe MnB bivalent rLP2086 composition was within 3% of the averageosmolality of the MenACWY-TT composition reconstituted with saline.

TABLE 7 Reconstituting Reading Reading Reading Average Vial Agent 1 mOsm2 mOsm 3 mOsm mOsm MenACWY- Saline 471 473 478 474 TT compositionMenACWY- MnB bivalent 487 487 489 488 TT rLP2086 composition composition

Example 12: Mening A, C, Y, and W-135 Polysaccharide ConjugateConcentrations in the Combined Drug Product

The concentration of the Mening A, C, Y and W-135-TT conjugates in theMenABCWY composition was assessed initially and again after 24 hours.The concentrations of the four conjugates were stable over the twentyfour hour time period (Table 8).

TABLE 8 Short Term Stability Results of the MnA, C, Y and W Conjugatesin the MenABCWY composition by ELISA MenACWY-TT composition + MnBbivalent The MenACWY-TT rLP2086 composition composition + Saline AfterAfter Sero- Initial, 24 hrs, Stability Initial, 24 hrs, Stability typeμg/mL μg/mL Ratio μg/mL μg/mL Ratio A 6.7 6.8 1.0 6.4 6.7 1.1 C 6.9 6.61.0 6.5 6.7 1.0 Y 8.1 8.7 1.1 9.6 9.8 1.0 W 8.5 8.8 1.0 8.8 9.0 1.0

Example 13: Evaluation of the Stability of the MnB Bivalent rLP2086Proteins in the MenABCWY Composition

Total and Bound rLP2086 Subfamily A (SEQ ID NO: 1) and Subfamily B (SEQID NO: 2) Protein Concentrations in the Combined Drug Product

The MenABCWY composition samples were analyzed by IEX-HPLC to determinethe protein concentrations. As shown in Table 9, the total protein,bound protein (to aluminum), and % bound of both MnB bivalent rLP2086Subfamily A (SEQ ID NO: 1) and Subfamily B (SEQ ID NO: 2) proteins(bound to aluminum) did not change within 24 hours indicating that therLP2086 Subfamily A and Subfamily B proteins were stable over the twentyfour hour time period.

TABLE 9 Total and Bound Protein Stability Bound Time Point, TotalProtein, Protein, Bound hours Subfamily μg/mL μg/mL Protein, %  0 A 8385 102 B 88 87  99 24 A 87 88 101 B 92 91  99

Example 14: rLP2086 Protein Purity and Peak Ratio in the CombinedMenABCWY Composition

The MenABCWY composition samples were analyzed by RP-HPLC to determinepurity and peak ratios for the rLP2086 proteins. See FIG. 3. The peak at11.9 min is excluded from the purity calculation.

rLP2086 Subfamily A and Subfamily B Protein IVRA in the Combined DrugProduct

The IVRA of the MenABCWY composition samples was assessed for up to 24hours after mixing. It was determined that the relative antigenicity ofthe rLP2086 Subfamily A (SEQ ID NO: 1) and Subfamily B (SEQ ID NO: 2)proteins in the MenABCWY composition was stable over the twenty fourhour time period.

Example 15: Mening A, C, Y, and W-135 Polysaccharide Stability in FullMenABCWY Composition Buffer Matrix by SEC-MALS

Stability of Mening a PS in Full MenABCWY Composition Buffer Matrix bySEC-MALLS after 6 and 24 Hours Incubation at Various Temperatures

Mening A, C, W, and Y polysaccharides were mixed with the Full MenABCWY

Composition Buffer Matrix and evaluated for stability by SEC-MALS afterincubation at 5° C., 25° C. and 37° C. for up to 24 hours. All fourpolysaccharides appear to be stable for up to 24 hours at 5° C. and 25°C. Some degradation was observed at 37° C. for Mening A and Y. Thedegree of degradation could not be determined for Mening YPolysaccharides due to formation of high Mw aggregates under all testedconditions except initial.

TABLE 10 Incubation Time, Incubation Sample hours Temperature Mw (kDa) ΔMw (%) Mening A 0 NA 169 N/A PS 24  5° C. 171 1 25° C. 157 −7 37° C. 126−26 Mening C 0 NA 213 N/A PS 24  5° C. 216 1 25° C. 215 1 37° C. 220 3Mening Y 0 NA 294 N/A PS* 24  5° C. 734 149 25° C. 756 157 37° C. 696136 Mening W- 0 NA 205 N/A 135 PS 24  5° C. 230 12 25° C. 211 3 37° C.219 7

Example 16: Evaluation of the Mnb Bivalent Rlp2086 Composition-InducedHsba Activity Cross Protection Against Menacwxy

The TRUMENBA MnB bivalent rLP2086 composition contains two lipidatedfactor H binding proteins (fHBP), one from each subfamily and providesprotection against Neisseria meningitides serogroup B. fHBP has beenfound to be expressed at variable levels in serogroups A, C, W, Y, and Xsuggesting that the MnB bivalent rLP2086 composition might offerprotection to these serogroups. We used sera from study B1971015 toinvestigate the proof of concept that the TRUMENBA MnB bivalent rLP2086composition may offer protection against serogroups A, C, W, Y, and X.Strains were collected globally (including MnW strains from a recent UKoutbreaks and MnX which is newly emerging in Africa). The process ofselecting a candidate strain to develop an hSBA and the immune responsefrom a subset of subjects immunized with the MnB bivalent rLP2086composition and a subset immunized with a four valent meningococcalcapsular polysaccharide conjugate vaccine (MCV4) as a comparison isdescribed.

Sera Used for Immunogenicity Determinations

Subsets of sera from the study B1971015, a phase 2, randomized,active-controlled, observer-blinded study in which two group of subjectsreceived either MENACTRA (meningococcal A, C, Y and W-135 polysaccharideconjugate vaccine [MCV4]) and ADACEL (tetanus toxoid, reduced diphtheriatoxoid, acellular pertussis vaccine [Tdap]) or bivalent rLP2086(TRUMENBA [meningococcal serogroup B vaccine], approved in the UnitedStates) were used to access the immunogenicity in hSBA using the MnACWYXstrains selected as described below.

Baseline Seropositivity Rates

Baseline seropositivity rates in the microcolony-based hSBA wereestimated using non-immune or prevaccination sera obtained fromadolescents from study B1971015. For this purpose, seropositivity isdefined as a hSBA titer ≥1:8, which was the anticipated assay LLOQ. Tobe considered for assay development, the strains must demonstrate lowbaseline seropositivity (i.e., a rate of hSBA titers ≥1:8 using baselinesera <˜40%).

Sera Used for Immunogenicity Determinations

Subsets of sera from the study B1971015, a phase 2, randomized,active-controlled, observer-blinded study in which two group of subjectsreceived either MENACTRA (meningococcal A, C, Y and W-135 polysaccharideconjugate vaccine [MCV4]) and ADACEL (tetanus toxoid, reduced diphtheriatoxoid, acellular pertussis vaccine [Tdap]) or bivalent rLP2086(TRUMENBA [meningococcal serogroup B vaccine], approved in the UnitedStates) were used to access the immunogenicity in hSBA using the MnACWYXstrains selected as described below.

Results and Discussion

Serogroup A

There were 17 strains from South Africa, 4 strains from US and onestrain from Netherlands in the Serogroup A SBA strain pool.Seventy-three percent of the serogroup A strains in this collectionexpress B16 and 23% B22. The prevalent clonal complexes in thiscollection of serogroup A strains are ST-1 and ST-5. Two strainsexpressing B16 and one strain expressing B22 with fHBP expression levelsabove 1100 WI were selected for further testing. 36 lots of C′ were usedfor C′ T30/T0 ratio screening. PMB1546 was excluded since appropriatehuman serum complement sources that did not non-specifically kill thestrain could not be identified according the C′ passing rate (Table 11).PMB3143 was excluded due to high baseline seropositivity rates, 52%,with adolescent preimmune sera. PMB3257 met the selection criteria:appropriate complement sources were identified, low baselineseropositivity rates with adolescent preimmune sera e.g. 0%, and hSBAswas technically feasible.

TABLE 11 Serogroup A Test Strain Candidates Baseline Epidemio- CountryC′ Sero- fHBP fHBP logical of Pass hSBA positivity Strain VariantExpression^(a) Marker Isolation Rate Compatible Rate PMB3257 B16 1725ST-1 South 33% Yes ^(b)  0% ^(c) complex/ Africa subgroup I/II PMB3143B16 1657 ST-1 U.S. 14% Yes 52%^(d) complex/ subgroup I/II PMB1546 B222657 ST-5 South  0% ND^(e) ND complex/ Africa subgroup III ^(a)meanfluorescent intensity as determined in the MEASURE assay ^(b)strainformed defined colonies, and was killed by indicator sera in hSBA^(c)adolescents, prevaccinated sera ^(d)pre-immunity rate high ^(e)notdone The selected Serogroup A strain is indicated in bold font.

Serogroup C

There were 49 strains from U. S. and 10 strains from Netherlands in theSerogroup C SBA strain pool. The fHBP sequence is more heterogeneous inserogroup C. The strain pool contains at least 17%, namely 19%, of MenCstrains were fHBP A10 expressing strains and 10% A15 expressing strains.The prevalent clonal complexes in this collection of serogroup C strainsare ST11/ET-37 complex and ST-103. One strain expresing A10 and threestrains epxresing Al 5 with fHBP expression levels above 1100 MFI wereselected for further testing. 36 lots of C′ were used for C′ T30/T0ratio screening. PMB5180, and PMB5196 expressing A15 for fHBP wereexcluded since appropriate human serum complement sources that did notnon-specifically kill the strain could not be identified according theC′ passing rate (Table 12). For PMB5043, the C′ passed initial screeningtest failed in SBA assay due to T30/T0 ratio. PMB5208 met the selectioncriteria: appropriate complement sources were identified, low baselineseropositivity rates with adolescent preimmune sera were confirmed 17%,and hSBAs was technically feasible.

TABLE 12 Serogroup C Test Strain Candidates fHBP Epidemio- Country C′Baseline fHBP Ex- logical of Pass hSBA Seropositivity Strain Variantpression^(a) marker Isolation Rate Compatible Rate PMB5208 A10 1563ST11/ United 56% Yes ^(b) 17% ^(d) ET-37 States complex PMB5180 A15 3237ST-103 United  0% NDe ND States PMB5196 A15 3353 ST-103 United  3% ND NDStates PMB5043 A15 2803 ST-103 United  8% No^(c) ND States ^(a)meanfluorescent intensity as determined in the MEASURE assay ^(b)strainformed defined colonies, and was killed by indicator sera, in hSBA^(c)no appropriate complement sources available, failed T30/T0 ratio inSBA ^(d)adolescents, prevaccinated sera ^(e)not done The selectedSerogroup C strain is indicated in bold font.

Serogroup W

There were 14 strains from U. S., 9 strains from Netherlands and 6recent outbreak strains from U. K. in the Serogroup W SBA strain pool.The fHBP sequence is more homogeneous in this collection of serogroup Wstrains. Forty-five percent of the strains in this pool expressed fHBPA19 variant and 45% fHBP A10. The prevalent clonal complex for fHBP Al 9variant strains in the serogroup W pool is ST-22 and the prevalentclonal complex for fHBP A10 variant strains is ST-11/ET-37 complex. Fromthe US, one strain expressing A19 and one strain expressing A10 wereselected. Two UK outbreak strains expressing A10 with fHBP expressionlevels above 1100 MFI were selected for further testing. 36 lots of C′were used for C′ T30/T0 ratio screening. Four strains all passed thisinitial C′ screening test. Based on the preimmunity rate, PMB5524 andPMB5163 were excluded since the seropositivity rates with adolescentpreimmune sera were 74% and 48%, respectively. PMB5248 and PMB5523 metthe selection criteria: appropriate complement sources were identified,low baseline seropositivity rates with adolescent preimmune sera e.g.26%, and 28%, respectively and hSBAs were technically feasible (Table13).

TABLE 13 Serogroup W Test Strain Candidates Epidemio- Country C′ hSBABaseline fHBP fHBP logical of Pass Com- Seropositivity Strain VariantExpression^(a) Marker Isolation Rate patible Rate PMB5248 A19 2684 ST-22United 14%   Yes ^(b) 26% ^(c) complex States PMB5524 A10 1197 ST-11/U.K. 34% Yes 74%^(d) ET-37 complex PMB5163 A10 1565 ST-11 United  6% Yes48%^(d) complex/ States ET-37 complex PMB5523 A10 1796 ST-11 U.K. 32%Yes 28%   complex/ ET-37 complex ^(a)mean fluorescent intensity asdetermined in the MEASURE assay ^(b)strain formed defined colonies, andwas killed by indicator sera, in hSBA ^(c)adolescents, prevaccinatedsera ^(d)pre-immunity rate high The selected Serogroup W strain isindicated in bold font.

Serogroup X

There were 8 strains from Africa and one strain from Netherlands in theSerogroup X SBA strain pool. Five strains from Africa express fHBP B49variant, three strains had a new fHBP type and the one strain fromNetherland expressed fHBP B09. The fHBP expression for all nine strainswas over 1100 MFI. Three Africa outbreak strains and one from theNetherlands were selected for further testing. 36 lots of C′ were usedfor C′ T30/T0 ratio screening. Four strains all passed C′ initialscreening test. Based on the preimmunity rate, PMB5467 was excludedsince the seropositivity rates with adolescent preimmune sera was 68%.PMB5537, PMB5540 and PMB5539 met the selection criteria: appropriatecomplement sources were identified, low baseline seropositivity rateswith adolescent preimmune sera e.g. 0, and hSBAs were technicallyfeasible (Table 14). PMB5540 was selected for evaluation.

TABLE 14 Serogroup X Test Strain Candidates Epidemio- Country C′ hSBABaseline fHBP fHBP logical of Pass Com- Seropositivity Strain VariantExpression^(a) Marker Isolation Rate patible Rate PMB5467 B09 1795ST-1157 Netherland  6% Yes^(b) 68%^(d) complex PMB5537  B239 4896 ST-181Burkina  8% Yes^(b)  4%^(c) complex Faso PMB5540 B49 8612 ST-181 Burkina53% Yes^(b)  0%   complex Faso PMB5539 B49 13706 To be Uganda 83%Yes^(b)  0%  assigned ^(a)mean fluorescent intensity as determined inthe MEASURE assay ^(b)strain formed defined colonies, and was killed byindicator sera in hSBA ^(c)no appropriate complement sources available,failed T30/10 ratio in SBA ^(c)adolescents, prevaccinated sera^(d)pre-immunity rate high The selected Serogroup X strain is indicatedin bold font.

Serogroup Y

There were 87 strains from U. S. and 30 strains from Netherlands in theSerogroup Y SBA strain pool. The fHBP sequence is more homogeneous inthis collection of serogroup Y strains. Sixty-six percent of theserogroup Y strains in the pool express fHBP A15. The common clonalcomplex for fHBP Al 5 variant expressing strains in this serogroup Ycollection is ST-23/Cluster A3. Only 15% of the serogroup Y strains inthis collection have fHBP expression levels above 1100 MFI. Threestrains from the A15 variant group with fHBP expression levels above1100 MFI were selected for further testing. 36 lots of C′ were used forC′ T30/T0 ratio screening. PMB5122, PMB5053 and PMB5050 were excludedsince appropriate human serum complement sources that did notnon-specifically kill the strain could not be identified according theC′ passing rate (Table 15). PMB5187 with fHBP B47 was selected for C′T30/T0 screening and C′ sources that did not kill the strainnon-specfically could be identified (Table 15). Even though PMB5187 didnot express the prevalent fHBP variant for serogroup Y, this strain metthe other selection criteria: appropriate complement sources wereidentified, low baseline seropositivity rates with adolescent preimmunesera e.g. 4%, and hSBAs was technically feasible.

TABLE 15 Serogroup Y Test Strain Candidates: Epidemio- Country C′ hSBABaseline fHBP fHBP logical of Pass Com- Seropositivity Strain VariantExpression^(a) Marker Isolation Rate patible Rate PMB5122 A15 1011 ST-United 0% ND^(d) ND 23/Cluster States A3 PMB5053 A15 1608 ST- United 0%ND ND 23/Cluster States A3 PMB5050 A15 1811 ST- United 0% ND ND23/Cluster States A3 PMB5187 B47 5063 ST- United 64% Yes ^(b) 4% ^(c)23/Cluster States A3 ^(a)mean fluorescent intensity as determined in theMEASURE assay ^(b)strain formed defined colonies, and was killed byindicator sera, in hSBA ^(c)adolescents, prevaccinated sera ^(d)not doneThe selected Serogroup Y strain is indicated in bold font.

MnACYWX strains were selected for hSBA development based on theprevalent variants of fHBP for the respective serogroup, fHBP expressionabove 1100 WI and assay feasibility (e.g. identification of humancomplement and baseline seropositivity). Characteristics of the sixselected MnACYWX strains, as well as the median fHBP expression for eachvariant group are summarized in Table 16.

TABLE 16 MnACYWX Test Strain Characteristics Sero- fHBP fHBP ClonalCountry of Strain group Variant Expression Complex Isolation PMB3257 AB16 1725 ST-1 complex South Africa PMB5208 C A10 1563 CC11/ET-37 UnitedStates complex PMB5248 W A19 2684 CC-22 United States PMB5523 W A10 1796CC-11/ET-37 U.K. complex PMB5187 Y B47 5063 CC-23/ United States ClusterA3 PMB5540 X B49 8612 ST-181 complex Burkina Faso

Immunogenicity Analysis

The MnACYWX test strains were used in hSBAs to assess the immuneresponse elicited in subsets of healthy adolescents aged 10 to <13 yearsenrolled in the Phase 2 concomitant study B1971015. TRUMENBA-elicitedhSBA responses were compared to MENACTRA-elicited (meningococcal A, C, Yand W-135 polysaccharide conjugate vaccine [MCV4]) hSBA responses forthe six strains selected (Table 17).

Substantial bactericidal antibody responses were observed in a highproportion of vaccinated individuals based on an hSBA titer ≥1:8, a morestringent criterion than the accepted correlate of protection (ie, anhSBA titer ≥1:4) for MenCWYX strains except MenA with 28% from thosesubjects immunized with TRUMENBA compared to 97% from those immunizedwith MCV4. The TRUMENBA response reached a peak at one month after dose2 for two serogroup W strains (PMB5248 100%, PMB5523, 97%) and one monthafter dose 3 for serogroup C, Y, X strains with response rates of83%-100%. MCV4 responses reached peak one month after dose 1, with noresponse for MenX. TRUMENBA elicited antibodies demonstrated thepotential to protect against MenX, which is not covered by MCV4. TheTRUMENBA response rate (i.e. %≥1:8) after 3 doses against MnC, W or Ystrains is comparable to MCV4 response rate after 1 dose. Theimmunogenicity data obtained from the MnACYWX hSBA test strains provideproof of concept for protection against serogroups other than B.

TABLE 17 The percentage of SBA titers ≥1:8 for MnACWYX strains VaccineGroup Group 2: Group 3: Trumenba MCV4 0-month 0, 2, 6-month StrainSerogroup Sampling Time N n % (GMT) N n % (GMT) PMB3257 A Month 0 30 13% (2) 30 1 3% (2) Month 1 30 29 97% (95) 30 4 13% (3)  Month 3 30 2273% (31) 30 6 20% (4)  Month 7 30 14 47% (11) 29 8 28% (5)  PMB5208 CMonth 0 30 6 20% (3)  30 7 23% (4)  Month 1 30 27  90% (119) 30 16 53%(8)  Month 3 30 28 93% (88) 30 21 70% (12) Month 7 30 27 90% (38) 30 2893% (29) PMB5248 W Month 0 30 12 40% (5)  30 8 27% (4)  Month 1 30 2997% (88) 30 22 73% (18) Month 3 29 28 97% (58) 30 30 100% (47)  Month 729 26 90% (41) 30 30 100% (77)  PMB5523 W Month 0 29 16 55% (8)  30 1343% (7)  Month 1 30 29 97% (60) 30 20 67% (15) Month 3 30 29 97% (58) 3029 97% (21) Month 7 30 29 97% (44) 30 30 100% (42)  PMB5187 Y Month 0 304 13% (3)  30 3 10% (3)  Month 1 30 29 97% (79) 30 14 47% (7)  Month 330 28 93% (58) 30 28 93% (31) Month 7 30 27 90% (36) 30 30 100% (58) PMB5540 X Month 0 30 0 0% (2) 30 2 7% (2) Month 1 30 0 0% (2) 30 5 17%(3)  Month 3 30 0 0% (2) 30 16 53% (7)  Month 7 30 0 0% (2) 30 25 83%(20)

CONCLUSION MnACYWX strains were selected for hSBA development based onthe prevalent variants of fHBP for the respective serogroup, fHBPexpression above the fHBP-expression threshold for MnB and assayfeasibility (e.g. identification of human complement and baselineseropositivity). Using sera from subjects immunized with Trumenba inhSBA using MnACYWX strains provided proof for the concept that Trumenbaelicited antibodies can provide protection against serogroups other thanB. Bivalent rLP2086-elicited responses (e.g. percentage of subjects withtiters ≥1:8) after 3 doses are comparable to MCV4-elicited responsesafter 1 dose for strains from serogroups C, W and Y, but lower for aserogroup A strain. Moreover, bivalent rLP2086-elicited hSBA titers ≥1:8in a high proportion of subjects, indicative of protection against MenX,which is not provided by MCV4.

Example 17: Trumenba Elicits Bactericidal Antibodies AgainstNon-Serogroup B Meningococci

Introduction. Neisseria meningitidis (Men) is the leading cause ofbacterial meningitis and septicemia in infants, adolescents, and youngadults. There are 5 major disease-causing meningococcal serogroups, A,B, C, Y, and W, with a sixth serogroup, X, emerging in Africa.Quadrivalent meningococcal conjugate vaccines (MCV4) are used to protectfrom disease caused by meningococcal serogroups A, C, Y, and W invarious regions of the world. The recently approved vaccine TRUMENBA®(MenB-FHbp, bivalent rLP2086, Pfizer Inc, Collegeville, Pa.), intendedto provide protection against serogroup B disease, consists of 2recombinant lipoproteins, 1 from each of the 2 factor H binding protein(FHbp) phylogenetic subfamilies. 2 The gene coding for FHbp is found innearly all invasive Men strains, independent of serogroupclassification. Preclinical studies have demonstrated the potential forMenB-FHbp to protect against other disease-causing serogroups. Inpreliminary studies, the Men B vaccine BEXSERO® (MenB-4C; Novartis Inc,Cambridge, Mass.), which includes a single FHbp variant antigen, wasable to elicit a bactericidal immune response against MenX and MenWstrains. The aim of this study was to extend these observations usingexploratory assays to investigate whether antibodies elicited inadolescents by MenB-FHbp are bactericidal against MenA, C, W, Y, and Xstrains (FIG. 7), thereby providing the potential for protection againstmeningococcal disease across serogroups.

Experimental Overview. From the candidate strains (FIG. 8), selectstrains that

-   -   Were not susceptible to human complement killing alone    -   Were killed in hSBA using sera shown to have bactericidal        antibodies directed against FHbp expressed by MenB strains    -   Have low baseline titers with prevaccination sera (25        prevaccination sera; see FIG. 9) For the selected strains (Table        18, FIG. 10): Develop exploratory hSBA assays. See FIG. 9 for        the sera tested in the hSBA. Response rate=percentage of        subjects with hSBA titers ≥1:8, greater than the established        correlate of protection (≥1:4) (FIG. 11-16).

TABLE 18 Characteristics of Selected MenA, MenC, MenY, MenW, and MenXTest Strains Median of fHBP fHBP Ex- Expression Sero- fHBP pression (WI)for Clonal Country of Strain group Variant (MFI) Sergroup ComplexIsolation PMB3257 A B16 1725 1192 ST-1 South complex/ Africa subgroupI/II PMB5208 C A10 1563 1563 ST-11 United complex/ States ET-37 complexPMB5248 W A19 2684 460 ST-22 United complex States PMB5523 W A10 1796460 ST-11 England/ complex/ Wales ET-37 complex PMB5187 Y B47 5063 290ST-23 United complex/ States cluster A3 PMB5540 X B49 8612 7555 ST-181Burkina complex Faso

With reference to FIG. 9, 25 sera from Group A month 0 were used tostudy pre-immunity. Sera from 30 subjects from Group A drawn at month 0and 1 and Group B drawn at month 0, 1, 3 and 7 were used for hSBAtesting. Endpoint of the response rate is the percentage of sera withtiters >1:8.

FIG. 10—Distribution of FHbp Surface Expression Levels (MFI) DeterminedFrom Flow Cytometric Experiments Using the FHbp Reactive mAb MN 994-11.The FHbp surface expression for each of the strains within a serogroupis noted with a black dot while the FHbp surface expression levels forthe selected test strains within each serogroup are noted with a coloredstar.

FIG. 11—hSBA Response Rate (Percentage of Subjects With hSBA Titers≥1:8) for MenA PMB3257 (B16). Response rates and 95% confidenceintervals for sera collected at preimmunization (month 0) and 1 monthafter doses 1, 2, and 3 for MenBFHbp are shown. The geometric meantiters (GMTs) obtained were 2, 3, 4, and 5, respectively. The responserates for subjects in the positive control group were 3% prior tovaccination and 97% one month after receiving MCV4. The GMTs for thepositive control group were 2 and 95, respectively.

FIG. 12—hSBA Response Rate (Percentage of Subjects With hSBA Titers≥1:8) for MenC PMB5208 (A10). Response rates and 95% confidenceintervals for sera collected at preimmunization (month 0) and 1 monthafter doses 1, 2, and 3 for MenBFHbp are shown. The GMTs obtained were4, 8, 12, and 29, respectively. The response rates for subjects in thepositive control group were 20% prior to vaccination and 90% one monthafter receiving MCV4. The GMTs for the positive control group were 3 and119, respectively.

FIG. 13—hSBA Response Rate (Percentage of Subjects With hSBA Titers≥1:8) for MenW PMB5248 (A19). Response rates and 95% confidenceintervals for sera collected at preimmunization (month 0) and 1 monthafter doses 1, 2, and 3 for MenBFHbp are shown. The GMTs obtained were4, 18, 47, and 77, respectively. The response rates for subjects in thepositive control group were 40% prior to vaccination and 97% one monthafter receiving MCV4. The GMTs for the positive control group were 5 and88, respectively.

FIG. 14—hSBA Response Rate (Percentage of Subjects With hSBA Titers≥1:8) for MenW PMB5523 (A10). Response rates and 95% confidenceintervals for sera collected at preimmunization (month 0) and 1 monthafter doses 1, 2, and 3 for MenBFHbp are shown. The GMTs obtained were7, 15, 21, and 42, respectively. The response rates for subjects in thepositive control group were 55% prior to vaccination and 97% one monthafter receiving MCV4. The GMTs for the positive control group were 8 and60, respectively.

FIG. 15—hSBA Response Rate (Percentage of Subjects With hSBA Titers≥1:8) for MenY PMB5187 (B47). Response rates and 95% confidenceintervals for sera collected at preimmunization (month 0) and 1 monthafter doses 1, 2, and 3 for MenBFHbp are shown. The GMTs obtained were3, 7, 31, and 58, respectively. The response rates for subjects in thepositive control group were 13% prior to vaccination and 97% one monthafter receiving MCV4. The GMTs for the positive control group were 3 and79, respectively.

FIG. 16—hSBA Response Rate (Percentage of Subjects With hSBA Titers≥1:8) for MenX PMB5540 (B49). Response rates and 95% confidenceintervals for sera collected at preimmunization (month 0) and 1 monthafter doses 1, 2, and 3 for MenBFHbp are shown. The GMTs obtained were2, 3, 7, and 20, respectively. The response rates for subjects in thepositive control group were 0% prior to vaccination and 0% one monthafter receiving MCV4 vaccine. The GMTs for the positive control groupwere 2 and 2, respectively.

Summary. Response rates elicited by MenB-FHbp peaked at 1 month afterdose 2 (Month 3) for the MenW strain, PMB5248 (100%), and 1 month afterdose 3 (Month 7) for the MenC, MenY, and MenX strains, as well as forthe second MenW strain (response rates ranging from 83%-100%).

The response rates measured by hSBA for the MenA test strain weresubstantially lower, peaking at 28% after 3 doses of MenB-FHbp.

The recognized correlate of protection against meningococcal disease isan hSBA titer ≥1:4. The ability of MenB-FHbp to elicit hSBA titers of atleast 1:8 provides proof of concept that MenB-FHbp may protect againstdisease caused by meningococcal serogroups other than B, including MenX,which is not covered by MCV4.

Example 18: A Phase 2, Randomized, Controlled, Observer-Blinded Study toDescribe the Immunogenicity, Safety, and Tolerability of Neisseriameningitidis Serogroup B Bivalent Recombinant Lipoprotein 2086 Vaccine(Bivalent rLP2086, i.e., now TRUMENBA® vaccine) in Healthy Subjects Aged≥24 Months to <10 Years (B1971017-syn)

Study Design: This was a Phase 2, randomized, controlled,observer-blinded, multicenter study designed to assess theimmunogenicity, safety, and tolerability of bivalent rLP2086 at the120-μg dose level administered to healthy subjects aged ≥24 months to<10 years as part of a Month 0, 2, and 6 schedule (Table 19).Approximately 400 subjects were planned to be randomly assigned to 1 of2 groups in a 3:1 ratio. Group 1 received bivalent rLP2086 at Month 0(Visit 1) followed by subsequent vaccinations at Months 2 and 6. Group 2received a licensed pediatric hepatitis A virus (HAV) vaccine at Month 0(Visit 1) and Month 6 and an injection with saline at Month 2 tomaintain the study blind. Follow-up visits were conducted 1 month aftereach vaccination and 6 months after the third vaccination to collectsafety data and/or obtain a blood sample. Subjects participated in thestudy for up to 13 months.

TABLE 19 Study Design Post- Month Post- Vaccina- Post- 12 Vaccina- tionVaccina- Follow- Vaccina- tion 1 Vaccina- 2 tion 3 up and tion Follow-tion Blood Vaccina- Blood Blood 1 up 2 Draw tion 3 Draw Draw Visit 1 2 34 5 6 7 Approximate 0 1 2 3 6 7 12 month Group 1 Bivalent BivalentBivalent (300 rLP2086 rLP2086 rLP2086 subjects) Group 2 HAV Saline HAV(100 vaccine vaccine subjects) Blood draw 5-10 mL 5-10 mL 5-10 mL 5-10mL (all subjects) HAV = hepatitis A virus

Vaccines Administered: Subjects in Group 1 were administered bivalentrLP2086 by intramuscular injection into the upper deltoid muscle of thearm at Months 0, 2, and 6. Subjects in Group 2 were administered HAVvaccine/saline/HAV vaccine into the upper deltoid muscle of the arm atMonths 0, 2, and 6, respectively.

Immunogenicity Evaluations: To facilitate immunogenicity analysis,subjects had approximately 5 to 10 mL (dependent upon age) of bloodcollected immediately before Vaccination 1, 1 month after Vaccination 2,and 1 and 6 months after Vaccination 3. For assessment of the immuneresponse to bivalent rLP2086, functional antibodies were analyzed invalidated hSBAs with 4 primary MnB test strains selected using anunbiased algorithm, and adjusted for epidemiological prevalence based onregulatory input, from Pfizer's MnB serum bactericidal assay (SBA)strain pool. The hSBA measures antibodies in human sera that initiatecomplement-dependent destruction of the target meningococcal strain.Four (4) primary MnB test strains, PMB80 (A22), PMB2001 (A56), PMB2948(B24), and PMB2707 (B44), each expressing an factor H binding protein(fHBP) variant heterologous to the vaccine component antigens, were usedin the hSBAs for determination of the immunogenicity endpoints in thisstudy. Sera obtained from all subjects prior to the first studyvaccination, 1 month after the second study vaccination, and 1 and 6months after the third study vaccination were used in these assays. Forthe primary analyses, 2 of the primary test strains (PMB80 [A22] andPMB2948 [B24]) were tested at each blood sampling time point for half ofthe subjects (in both groups), and the other 2 primary test strains(PMB2001 [A56] and PMB2707 [B44]) were tested at each blood samplingtime point for the remaining half of the subjects. The primaryimmunogenicity endpoints were: Proportion of subjects aged ≥24 months to<4 years (at study entry) with hSBA titer ≥lower limit of quantitation(LLOQ) for each of the 4 primary MnB test strains 1 month after thethird vaccination with bivalent rLP2086; and Proportion of subjects aged≥4 years to <10 years (at study entry) with hSBA titer ≥LLOQ for each ofthe 4 primary MnB test strains 1 month after the third vaccination withbivalent rLP2086.

The secondary immunogenicity endpoints were:

-   -   In healthy subjects aged ≥24 months to <10 years at study entry:        -   Proportion of subjects with hSBA titer ≥LLOQ for each of the            4 primary MnB test strains 1 month after the third            vaccination with bivalent rLP2086.    -   In healthy subjects aged ≥24 months to <4 years at study entry,        in healthy subjects aged ≥4 years to <10 years at study entry,        and in the combined age stratum:        -   Proportion of subjects with hSBA titer ≥LLOQ for each of the            4 primary MnB test strains 1 month after the second            vaccination and 1 and 6 months after the third vaccination            with bivalent rLP2086.        -   Proportions of subjects achieving hSBA titers of ≥1:4, ≥1:8,            ≥1:16, ≥1:32, ≥1:64, and ≥1:128 for each of the 4 primary            test strains at baseline, 1 month after the second            vaccination, and 1 and 6 months after the third vaccination            with bivalent rLP2086.        -   hSBA GMTs for each of the 4 primary test strains at            baseline, 1 month after the second vaccination, and 1 and 6            months after the third vaccination with bivalent rLP2086.

The secondary immunogenicity endpoints were summarized for both theevaluable immunogenicity population and the mITT population.

The following exploratory endpoints were used to describe responses inhealthy subjects aged ≥24 months to <4 years at study entry, in healthysubjects aged ≥4 years to <10 years at study entry, and in the combinedage stratum:

-   -   Proportions of subjects with hSBA titers ≥1:4, ≥1:8, ≥1:16,        ≥1:32, ≥1:64, and ≥1:128 at each applicable blood sampling time        point.    -   hSBA GMTs for each of the 4 primary strains at each applicable        blood sampling time point.    -   Proportion of subjects achieving at least a 4-fold increase in        hSBA titer from baseline to 1 month after the third vaccination        with bivalent rLP2086 for each of the 4 primary test strains:        -   For subjects with a baseline hSBA titer below the limit of            detection (LOD) or an hSBA titer of <1:4, a 4-fold response            was defined as an hSBA titer of ≥1:16 or the LLOQ (whichever            titer was higher).        -   For subjects with a baseline hSBA titer of ≥LOD (ie, hSBA            titer of ≥1:4) and <LLOQ, a 4-fold response was defined as            an hSBA titer of ≥4 times the LLOQ.        -   For subjects with a baseline hSBA titer of ≥LLOQ, a 4-fold            response was defined as an hSBA titer of ≥4 times the            baseline titer.

Results

Subjects: A total of 400 subjects aged ≥24 months to <10 years wererandomized in this study. Of the subjects randomized, 294 subjects werein Group 1 (bivalent rLP2086) and 106 subjects were in Group 2(HAV/saline). There were 200 subjects randomized in each of the≥24-month to <4-year and ≥4-year to <10-year age strata.

Of the 400 randomized subjects, 390 (97.5%) subjects completed thevaccination phase (through 1 month after last study vaccination) of thestudy. A total of 387 (96.8%) subjects completed the 6-month follow-uptelephone contact. Only subjects who completed the vaccination phase andthe 6-month follow-up telephone contact were considered to havecompleted the study. Overall, a total of 375 (93.8%) subjects completedall study procedures and completion was similar in each age strata. Atotal of 371 (92.8%) subjects were included in the evaluableimmunogenicity population, and 29 (7.3%) subjects were excluded from theevaluable immunogenicity population. All 400 randomized subjects wereincluded in the mITT population.

Immunogenicity Results: The primary objectives of this study were todescribe subject immune response to bivalent rLP2086 as measured by hSBAagainst 4 primary MnB test strains, 2 expressing an LP2086 subfamily Aprotein and 2 expressing an LP2086 subfamily B protein, measured 1 monthafter the third vaccination in healthy subjects aged ≥24 months to <4years and ≥4 years to <10 years. The description of immune responses forthe combined age stratum (≥24 months to <10 years) was a secondaryobjective. The endpoints for the primary objectives were the proportionsof subjects in each age stratum achieving hSBA titers ≥LLOQ for each ofthe 4 primary MnB strains 1 month after the third vaccination.

A robust immune response was observed for children aged ≥24 months to<10 years 1 month after the third dose of bivalent rLP2086, as confirmedby the proportion of subjects achieving an hSBA titer ≥LLOQ (1:8 forA56, B24 and B44; 1:16 for A22) for each of the 4 primary MnB teststrains ranging from 80.0% to 100.0% for subjects aged ≥24 months to <4years and from 78.3% to 100.0% for subjects ≥4 years to <10 years after3 doses. The proportion of subjects in the combined age stratum with anhSBA titer ≥LLOQ for each of the 4 primary MnB test strains 1 monthafter the third vaccination ranged from 79.1% to 100.0%. These findingsare further supported by substantial GMTs (range 19.1 to 191) and in theproportion of subjects achieving an hSBA titer ≥1:4 (81.5% to 100%) or≥1:16 (75.4% to 100%) against each of the 4 primary MnB test strainsafter 3 doses of bivalent rLP2086 compared to baseline across both agestrata. Additionally, the proportion of subjects in the combined agestratum achieving an hSBA fold rise ≥4 from baseline to 1 month afterthe third vaccination for each of the 4 primary MnB test strains rangedfrom 76.9% to 93.5%.

The secondary objective of the study was to describe immune responses 1month after the second dose of bivalent rLP2086, as assessed by ≥LLOQresponses, defined hSBA titers and hSBA GMTs for the 2 age strata andthe combined age stratum. For the combined age stratum, the proportionof subjects achieving an hSBA titer ≥LLOQ ranged from 48.5% to 100.0%with no meaningful differences observed between the younger and olderage strata. These findings are further supported by the combined agestratum with increases in GMTs (range 11.1 to 96.6) and in theproportion of subjects achieving an hSBA titer ≥1:4 (57.7% to 100%) or≥1:16 (43.1% to 100%) after 2 doses of bivalent rLP2086 compared tobaseline against each of the 4 primary MnB test strains. GMTs weresimilar between the 2 age strata. Additionally, the proportion ofsubjects in the combined age stratum achieving an hSBA fold rise ≥4 frombaseline to 1 month after the second vaccination for each of the 4primary MnB test strains ranged from 42.3% to 91.0%.

Immunopersistence was also assessed at 6 months after the third dose ofbivalent rLP2086 with the proportion of subjects with an hSBA titer≥LLOQ declining from 79.1% to 100% 1 month after Vaccination 3 to 10.4%to 82.4% at 6 months after the third vaccination for the combined agestratum. No differences between the 2 age strata were observed exceptfor A22, for which older children had a higher proportion of subjectsachieving a titer ≥LLOQ than the younger children (46%, 95% Cl 33.4,59.1 vs 19%, 95% CI 10.2, 30.9). However, baseline prevaccination ratesof titers ≥LLOQ were greater for A22 in the older age stratum (13.6% vs4.4%). A similar trend was also observed for the combined age stratumfor the proportion of subjects with a protective hSBA titer ≥1:4,ranging from 13.3% to 84.0% and GMTs, ranging from 5.1 to 31.3 at 6months after the third vaccination.

In summary, bivalent rLP2086 given as 3 doses on a 0-, 2-, and 6-monthschedule elicits a robust immune response among toddlers and childrenaged ≥24 months to <10 years with protective antibody titers achieved asmeasured by hSBA in a high proportion of subjects after the third dose.No clinically meaningful differences were observed between toddlers aged≥24 months to <4 years and children aged ≥4 years to <10 years. Antibodyresponses decline 6 months after the third dose, but remain higher thanprevaccination baseline rates.

Conclusion(s): In conclusion, bivalent rLP2086 administered to toddlersand children aged ≥24 months to <10 years in a 3-dose series on a 0-,2-, and 6-month schedule elicits a robust immune response by themajority of subjects after the second and third doses, with protectiveantibody titers achieved after the third dose as measured by hSBAs. hSBAtiters decreased 6 months after a 3-dose series. The vaccine, asadministered in this study, was safe and well tolerated with anacceptable safety profile for toddlers and children aged ≥24 months to<10 years.

EXAMPLE 19: A Phase 2, Randomized, Controlled, Observer-Blinded Study toDescribe the Immunogenicity, Safety, and Tolerability of Neisseriameningitidis Serogroup B Bivalent Recombinant Lipoprotein 2086 Vaccine(Bivalent rLP2086, i.e., now TRUMENBA@ vaccine) in Healthy Subjects Aged≥24 Months to <10 Years (B1971017-CSR)

The initial formulation of bivalent rLP2086 (which did not includepolysorbate-80) has been assessed in Phase 1 studies in adults,adolescents, children, and toddlers with satisfactory safety,tolerability, and immunogenicity profiles demonstrated in thesepopulations. The initial formulation has been shown to have anacceptable safety profile up to a dose of 200 μg, and to be immunogenicas measured by hSBA. In toddlers aged 18 to 36 months, the initialformulation has been studied at dose levels of 20 μg, 60 μg, and 200 μg(Study 6108A1-502-AU). In the 6108A1-502-AU study, frequencies of localreactions in each dose group were generally higher than those in thehepatitis A virus (HAV) vaccine/placebo group, but in most cases thereactions were of mild or moderate severity. The incidence of fevertended to increase with increasing dose level. The proportions ofsubjects reporting any fever after any dose were 36.4% in the 20-μggroup, 39.1% in the 60-μg group, and 54.5% in the 200-μg group. Thefrequencies of other systemic events in each vaccine group weregenerally comparable to those for HAV/placebo. The majority of subjectshad an hSBA response for the MnB strains following the thirdvaccination.

Compared to the initial formulation, the drug substance manufacturingprocess and the drug product formulation have undergone enhancements(including the addition of polysorbate-80) designed to increasescalability for manufacture and to ensure long-term stability of thefinal formulation of the vaccine. Safety data from adults andadolescents participating in studies with the final formulation ofbivalent rLP2086 are consistent with the safety of the initialformulation. Local reactions and systemic events were generally mild ormoderate in severity in all age groups. Severe events were relativelyinfrequent. Furthermore, follow-up adverse event (AE) data fromimmunopersistence studies 6108A1-1002-AU, 6108A1-2001, and B1971033ranging from 6 months to 48 months after the third dose of bivalentrLP2086 raised no safety concerns. Serious adverse events (SAEs) wereinfrequent, and mostly considered not related to the study vaccine.There were few withdrawals from the studies due to AEs.

Two (2) Phase 2 studies (B1971017 and B1971035) were conducted toexplore the immunogenicity and safety of the vaccine in children (12months to <10 years) with the final formulation of the vaccine. StudyB1971035 is ongoing and designed to assess the safety, tolerability, andimmunogenicity of 2 different dose levels (60-μg and 120-μg) amonghealthy toddlers aged 12 months to <24 months. This study (B1971017)assessed the immunogenicity, safety, and tolerability of bivalentrLP2086 at the 120-μg dose level (final formulation) administered tohealthy subjects aged ≥24 months to <10 years as part of a Month 0, 2,and 6 schedule. Approximately 400 subjects were planned to be randomizedto 1 of 2 groups in a 3:1 ratio. Group 1 received bivalent rLP2086 atMonth 0 (Visit 1) followed by subsequent vaccinations at Months 2 and 6.Group 2 received HAV vaccine at Month 0 (Visit 1) and Month 6 and aninjection with saline at Month 2. Randomization was stratified to ensurethat equal numbers of subjects were included in the ≥24-month to <4-yearand ≥4-year to <10-year age strata. This (B1971017) was a Phase 2,randomized, controlled, observer-blinded, multicenter study in whichapproximately 400 subjects were planned to be randomly assigned to 1 of2 groups in a 3:1 ratio. Group 1 received bivalent rLP2086 at Month 0(Visit 1) followed by subsequent vaccinations at Months 2 and 6. Group 2received a licensed pediatric HAV vaccine at Month 0 (Visit 1) and Month6 and an injection with saline at Month 2 to maintain the study blind.Randomization was stratified according to age to ensure that equalnumbers of subjects were included in the ≥24-month to <4-year agestratum and the ≥4-year to <10-year age stratum. The study was plannedto enroll approximately 200 subjects aged ≥24 months to <4 years andapproximately 200 subjects aged ≥4 years to <10 years.

This study assessed the immunogenicity, safety, and tolerability ofbivalent rLP2086 at the 120-μg dose level administered to healthysubjects aged ≥24 months to <10 years as part of a Month 0, 2, and 6schedule. Follow-up visits were conducted 1 month after each vaccinationand 6 months after the third vaccination to collect safety data and/orobtain a blood sample. Subjects participated in the study for up to 13months. Bivalent rLP2086 (containing 60 μg each of a purified subfamilyA and subfamily B rLP2086 protein, adsorbed to aluminum in a sterilebuffered isotonic suspension) was provided in a 0.5-mL dose forinjection.

A licensed pediatric HAV vaccine was provided in a 0.5-mL dose forinjection. For Group 1, 59.31% of subjects aged ≥24 months to <4 yearsand 57.05% of subjects aged ≥4 years to <10 years received concomitanttreatment. For Group 2, 58.18% of subjects aged ≥24 months to <4 yearsand 52.94% of subjects aged ≥4 years to <10 years received concomitanttreatment. The most common concomitant treatments received during thestudy were ibuprofen, paracetamol, and amoxicillin.

Bivalent rLP2086 Serum Bactericidal Assay—Primary Test Strains

For assessment of the immune response to bivalent rLP2086, functionalantibodies were analyzed in validated hSBAs with 4 primary MnB teststrains selected using an unbiased algorithm, and adjusted forepidemiological prevalence based on regulatory input, from Pfizer's MnBserum bactericidal assay (SBA) strain pool. The hSBA measures antibodiesin human sera that initiate complement-dependent destruction of thetarget meningococcal strain. Four (4) primary MnB test strains, PMB80(A22), PMB2001 (A56), PMB2948 (B24), and PMB2707 (B44), each expressingan fHBP variant heterologous to the vaccine component antigens, wereused in the hSBAs for determination of the immunogenicity endpoints inthis study. Sera obtained from all subjects prior to the first studyvaccination, 1 month after the second study vaccination, and 1 and 6months after the third study vaccination were used in these assays.

For the primary analyses, 2 of the primary test strains (PMB80 [A22] andPMB2948 [B24]) were tested at each blood sampling time point for half ofthe subjects (in both groups), and the other 2 test primary strains(PMB2001 [A56] and PMB2707 [B44]) were tested at each blood samplingtime point for the remaining half of the subjects.

Immunogenicity Analysis

There was no hypotheses testing for immunogenicity analysis. Anestimation approach was used to assess the primary, secondary, andexploratory objectives.

The proportions of subjects in each group achieving hSBA titer ≥lowerlimit of quantitation (LLOQ) 1 month after the second and thirdvaccination and 6 months after the third vaccination were computed foreach test strain, along with 2-sided 95% exact confidence intervals(Cls), for each of the age strata and the combined age stratum.

Primary Immunogenicity Endpoints

The primary immunogenicity endpoints were:

-   -   Proportion of subjects aged ≥24 months to <4 years (at study        entry) with hSBA titer ≥LLOQ for each of the 4 primary MnB test        strains 1 month after the third vaccination with bivalent        rLP2086.    -   Proportion of subjects aged ≥4 years to <10 years (at study        entry) with hSBA titer ≥LLOQ for each of the 4 primary MnB test        strains 1 month after the third vaccination with bivalent        rLP2086.

Secondary Immunogenicity Endpoints

The secondary immunogenicity endpoints were:

-   -   In healthy subjects aged ≥24 months to <10 years at study entry:        -   Proportion of subjects with hSBA titer ≥LLOQ for each of the            4 primary MnB test strains 1 month after the third            vaccination with bivalent rLP2086.    -   In healthy subjects aged ≥24 months to <4 years at study entry,        in healthy subjects aged ≥4 years to <10 years at study entry,        and in the combined age stratum:        -   Proportion of subjects with hSBA titer ≥LLOQ for each of the            4 primary MnB test strains 1 month after the second            vaccination and 1 and 6 months after the third vaccination            with bivalent rLP2086.        -   Proportions of subjects achieving hSBA titers of ≥1:4, ≥1:8,            ≥1:16, ≥1:32, ≥1:64, and ≥1:128 for each of the 4 primary            test strains at baseline, 1 month after the second            vaccination, and 1 and 6 months after the third vaccination            with bivalent rLP2086.        -   hSBA GMTs for each of the 4 primary test strains at            baseline, 1 month after the second vaccination, and 1 and 6            months after the third vaccination with bivalent rLP2086.

Exploratory Immunogenicity Endpoints

For exploratory endpoints, testing was not performed on all 4 primaryMnB test strains. Instead, 50% of subjects were tested using strainsPMB2001 (A56) and PMB2707 (B44), but not PMB80 (A22) or PMB2948 (B24).The remaining 50% of subjects were tested using strains PMB80 (A22) orPMB2948 (B24), but not PMB2001 (A56) or PMB2707 (B44). All of theexploratory endpoints specified below may have been applied to hSBAresults from all subjects who received bivalent rLP2086 and may havebeen tested for the appropriate strain at the indicated time point(s).

The following exploratory endpoints were used to describe responses inhealthy subjects aged ≥24 months to <4 years at study entry, in healthysubjects aged ≥4 years to <10 years at study entry, and in the combinedage stratum:

-   -   Proportions of subjects with hSBA titers ≥1:4, ≥1:8, ≥1:16,        ≥1:32, ≥1:64, and ≥1:128 at each applicable blood sampling time        point.    -   hSBA GMTs for each of the 4 primary strains at each applicable        blood sampling time point.    -   Proportion of subjects achieving at least a 4-fold increase in        hSBA titer from baseline to 1 month after the third vaccination        with bivalent rLP2086 for each of the 4 primary test strains:        -   For subjects with a baseline hSBA titer below the limit of            detection (LOD) or an hSBA titer of <1:4, a 4-fold response            was defined as an hSBA titer of ≥1:16 or the LLOQ (whichever            titer was higher).        -   For subjects with a baseline hSBA titer of ≥LOD (ie, hSBA            titer of ≥1:4) and <LLOQ, a 4-fold response was defined as            an hSBA titer of ≥4 times the LLOQ.        -   For subjects with a baseline hSBA titer of ≥LLOQ, a 4-fold            response was defined as an hSBA titer of ≥4 times the            baseline titer.

Methods of Analysis

The primary analysis for immunogenicity included an estimate for theproportion of subjects in each group achieving an hSBA titer ≥LLOQ 1month after the third vaccination for each test strain, along with2-sided 95% exact confidence Cls, for each of the age strata and in thecombined age stratum.

All of the binary endpoints (including primary endpoints) weresummarized with 2-sided 95% CIs using the exact method. GMTs on hSBAresults were also summarized with 95% CIs.

The LLOQ was 1:16 for PMB80 (A22), 1:8 for PMB2001 (A56), 1:8 forPMB2707 (B44), and 1:8 for PMB2948 (B24).

For the calculation of GMTs, hSBA results below the LLOQ were set as0.5×LLOQ for the primary analysis.

Analysis of Primary Endpoints

The primary analysis for the primary objectives was based on theevaluable immunogenicity population. The proportion of subjects in eachgroup achieving hSBA titer ≥LLOQ 1 month after the third vaccination wascomputed for each test strain with 2-sided 95% exact Cls. To address the2 primary objectives, these data are presented for the 2 age strata: ≥24months to <4 years and ≥4 years to <10 years.

All of the binary endpoints (including primary endpoints) weresummarized with 2-sided 95% CIs using the exact method. GMTs on hSBAresults were also summarized with 95% CIs.

To support the interpretation of the primary analyses, an identicalanalysis based on the mITT population was conducted.

Analysis of Secondary and Exploratory Endpoints

The following analyses addressed the secondary and exploratoryimmunogenicity objectives:

-   -   The proportions of subjects achieving hSBA titers ≥LLOQ for each        of the 4 primary strains at 1 month after the second vaccination        and 6 months after the third vaccination were analyzed in the 2        age strata separately and combined, in the evaluable        immunogenicity and the mITT populations.    -   The proportions of subjects achieving hSBA titers of ≥1:4, ≥1:8,        ≥1:16, ≥1:32, ≥1:64, and ≥1:128 for each of the 4 primary test        strains at baseline, 1 month after the second vaccination, and 1        and 6 months after the third vaccination were analyzed in the 2        age strata separately and combined, in the evaluable        immunogenicity and the mITT populations.    -   The hSBA GMTs for each of the 4 primary test strains at        baseline, 1 month after the second vaccination, and 1 and 6        months after the third vaccination were analyzed in the 2 age        strata separately and combined, in the evaluable immunogenicity        and the mITT populations.

The exploratory endpoints were summarized, in the 2 age strataseparately and combined, for each applicable time point for both theevaluable immunogenicity population and the mITT population.

Reverse Cumulative Distribution Curves

The empirical reverse cumulative distribution curves (RCDCs) wereassessed graphically for each of the 4 primary strains and at eachsampling time point, for the evaluable immunogenicity population.

Immunogenicity Evaluation

Populations Analyzed

The evaluable immunogenicity population was the primary analysispopulation for the immunogenicity analyses. The mITT population was usedas a supportive immunogenicity population for the immunogenicityanalyses.

A total of 371 (92.8%) subjects were included in the evaluableimmunogenicity population, and 29 (7.3%) subjects were excluded from theevaluable immunogenicity population. Subjects could have been excludedfrom the immunogenicity populations for more than 1 reason. A total of21 (5.3%) subjects were excluded from the evaluable immunogenicitypopulation because they did not have baseline blood drawn prior to thefirst dose of vaccine or after Vaccination 3, 15 (3.8%) subjects did nothave a valid and determinate assay result at any visit, 11 (2.8%)subjects were not eligible or became ineligible for the study before orat the 1-month post-Vaccination 3 visit, 11 (2.8%) subjects did notreceive vaccine as randomized at all vaccination visits, and 4 (1.0%)subjects had an important protocol deviation as identified by themedical monitor. Overall, the 2 study groups and 2 age strata werecomparable with respect to the percentages of subjects who were excludedfrom the evaluable immunogenicity population.

All 400 randomized subjects were included in the mITT population.

Immunogenicity Results

The results of the analyses for the primary immunogenicity endpoints,secondary immunogenicity endpoints, and exploratory immunogenicityendpoints are provided in the following sections.

Primary and Secondary Endpoints

Proportion of Subjects Achieving an hSBA Titer LLOQ

The primary immunogenicity endpoints were the proportion of subjectsaged ≥24 months to <4 years (at study entry), and aged ≥4 years to <10years (at study entry), with an hSBA titer ≥LLOQ for each of the 4primary MnB test strains 1 month after the third vaccination withbivalent rLP2086. The proportion of all subjects in the combined agestratum (at study entry) with an hSBA titer ≥LLOQ for each of the 4primary MnB test strains 1 month after the third vaccination withbivalent rLP2086, along with the proportion of subjects in theindividual and combined age strata with an hSBA titer ≥LLOQ for each ofthe 4 primary MnB test strains 1 month after the second vaccination withbivalent rLP2086, were secondary endpoints.

The proportion of subjects in each age stratum with an hSBA titer ≥LLOQfor each of the 4 primary MnB test strains is presented in Table 20 forthe evaluable immunogenicity population.

The proportion of subjects aged ≥24 months to <4 years and ≥4 years to<10 years in Group 1 with an hSBA titer ≥LLOQ at baseline was 4.4% and13.6%, respectively, for PMB80 (A22); 1.5% and 15.4%, respectively, forPMB2001 (A56); 3.0% and 7.5%, respectively, for PMB2948 (B24); and 0.0%,for both age strata for PMB2707 (B44). Overall, the proportion ofsubjects in the combined age stratum with an hSBA titer ≥LLOQ atbaseline was 9.0% for PMB80 (A22), 8.3% for PMB2001 (A56), 5.2% forPMB2948 (B24), and 0.0% for PMB2707 (B44) in Group 1.

The proportion of subjects aged ≥24 months to <4 years and ≥4 years to<10 years in Group 1 with an hSBA titer >LLOQ at 1 month after thesecond vaccination was 59.4% and 78.8%, respectively, for PMB80 (A22);100.0% for both age strata for PMB2001 (A56); 49.2% and 65.1%,respectively, for PMB2948 (B24); and 57.1% and 40.3%, respectively, forPMB2707 (B44).

Overall, the proportion of subjects in the combined age stratum with anhSBA titer ≥LLOQ at 1 month after the second vaccination was 69.2% forPMB80 (A22), 100.0% for PMB2001 (A56), 57.0% for PMB2948 (B24), and48.5% for PMB2707 (B44) in Group 1.

The proportion of subjects aged ≥24 months to <4 years and ≥4 years to<10 years in Group 1 with an hSBA titer >LLOQ at 1 month after the thirdvaccination was 83.8% and 91.0%, respectively, for PMB80 (A22); 100.0%for both age strata for PMB2001 (A56); 85.7% and 92.1%, respectively,for PMB2948 (B24); and 80.0% and 78.3%, respectively, for PMB2707 (B44).

Overall, the proportion of subjects in the combined age stratum with anhSBA titer ≥LLOQ at 1 month after the third vaccination was 87.4% forPMB80 (A22), 100.0% for PMB2001 (A56), 88.9% for PMB2948 (B24), and79.1% for PMB2707 (B44) in Group 1. In general, the proportion of Group2 subjects with an hSBA titer >LLOQ did not change overtime compared tobaseline. The proportion of subjects in the combined age stratum with anhSBA titer ≥LLOQ at any time point ranged from 4.4% to 8.5% for PMB80(A22); 14.9% to 20.9% for PMB2001 (A56); 0.0% to 8.9% for PMB2948 (B24);and 0.0% at each time point for PMB2707 (B44).

Results for the mITT population were similar to those of the evaluableimmunogenicity population.

Subgroup analyses of the proportion of subjects with an hSBA titer ≥LLOQfor each of the 4 primary MnB test strains are presented for theevaluable immunogenicity population by sex, race, and country. Therewere no clinically important differences observed in the subgroupanalyses performed.

Immunopersistence: Proportion of Subjects Achieving hSBA Titer ≥LLOQ 6Months after Third Vaccination

The proportion of subjects aged ≥24 months to <4 years, ≥4 years to <10years, and in the combined age stratum (≥24 months to <10 years), withan hSBA titer ≥LLOQ for each of the 4 primary MnB test strains 6 monthsafter the third vaccination with bivalent rLP2086 was a secondaryimmunogenicity endpoint. The proportion of subjects with an hSBAtiter >LLOQ for each of the 4 primary MnB test strains in each agestratum is presented in Table 20 for the evaluable immunogenicitypopulation.

In general, there was a decline in the proportion of subjects with anhSBA titer ≥LLOQ for each of the 4 primary MnB test strains observedamong Group 1 subjects in both age strata at 6 months after the thirdvaccination.

For subjects aged ≥24 months to <4 years, from 1 month after the thirdvaccination to 6 months after the third vaccination, the proportion ofsubjects with an hSBA titer ≥LLOQ decreased from 83.8% to 19.0%,respectively, for PMB80 (A22); 100.0% to 80.3%, respectively, forPMB2001 (A56); 85.7% to 9.2%, respectively for PMB2948 (B24); and 80.0%to 12.1%, respectively, for PMB2707 (B44).

For subjects aged ≥4 years to <10 years, from 1 month after the thirdvaccination to 6 months after the third vaccination, the proportion ofsubjects with an hSBA titer ≥LLOQ decreased from 91.0% to 46.0%,respectively, for PMB80 (A22); 100.0% to 84.3%, respectively, forPMB2001 (A56); 92.1% to 21.9%, respectively for PMB2948 (B24); and 78.3%to 8.7%, respectively, for PMB2707 (B44).

Overall for the combined age stratum, from 1 month after the thirdvaccination to 6 months after the third vaccination, the proportion ofsubjects with an hSBA titer >LLOQ decreased from 87.4% to 32.5% forPMB80 (A22); 100.0% to 82.4% for PMB2001 (A56); 88.9% to 15.5% forPMB2948 (B24); and 79.1% to 10.4% for PMB2707 (B44).

In general, the proportion of Group 2 subjects with an hSBA titer >LLOQdid not change overtime compared to baseline.

TABLE 20 Subjects With hSBA Titer ≥LLOQ for Primary Strains— EvaluableImmunogenicity Population Strain (Variant) Vaccine Group (as Randomized)Sampling Time Point Group 1 rLP2086 Group 2 HAV/Saline Age Strata N^(a)n^(b) (%) (95% CI)^(c) N^(a) n^(b) (%) (95% CI)^(c) PMB80 (A22) BeforeVaccination 1 ≥24 Months to <10 years 134 12 (9.0)  (4.7, 15.1) 47 3(6.4) (1.3, 17.5) ≥24 Months to <4 years 68 3 (4.4)  (0.9, 12.4) 26 1(3.8) (0.1, 19.6) ≥4 Years to <10 years 66 9 (13.6)  (6.4, 24.3) 21 2(9.5) (1.2, 30.4) 1 Month after Vaccination 2 ≥24 Months to <10 years130 90 (69.2) (60.5, 77.0) 45 2 (4.4) (0.5, 15.1) ≥24 Months to <4 years64 38 (59.4) (46.4, 71.5) 24 0 (0.0) (0.0, 14.2) ≥4 Years to <10years 6652 (78.8) (67.0, 87.9) 21 2 (9.5) (1.2, 30.4) 1 Month after Vaccination3 ≥24 Months to <10 years 135 118 (87.4) (80.6, 92.5) 45 3 (6.7) (1.4,18.3) ≥24 Months to <10 years 68 57 (83.8) (72.9, 91.6) 25 1 (4.0) (0.1,20.4) ≥24 Months to <10 years 67 61 (91.0) (81.5, 96.6) 20 2 (10.0)(1.2, 31.7) 6 Months after Vaccination 3 ≥24 Months to <10 years 126 41(32.5) (24.5, 41.5) 47 4 (8.5) (2.4, 20.4) ≥24 Months to <4 years 63 12(19.0) (10.2, 30.9) 26 2 (7.7) (0.9, 25.1) ≥4 Years to <10 years 63 29(46.0) (33.4, 59.1) 21 2 (9.5) (1.2, 30.4) PMB2001 (A56) BeforeVaccination 1 ≥24 Months to <10 years 132 11 (8.3)  (4.2, 14.4) 47 7(14.9) (6.2, 28.3) ≥24 Months to <4 years 67 1 (1.5) (0.0, 8.0) 24 2(8.3) (1.0, 27.0) ≥4 Years to <10 years 65 10 (15.4)  (7.6, 26.5) 23 5(21.7) (7.5, 43.7) 1 Month after Vaccination 2 ≥24 Months to <10 years43 7 (16.3)  (6.8, 30.7) 133 133 (100.0) (97.3, 100.0) ≥24 Months to <4years 66 66 (100.0)  (94.6, 100.0) 21 2 (9.5) (1.2, 30.4) ≥4 Years to<10 years 67 67 (100.0)  (94.6, 100.0) 22 5 (22.7) (7.8, 45.4) 1 Monthafter Vaccination 3 ≥24 Months to <10 years 139 139 (100.0)  (97.4,100.0) 43 9 (20.9) (10.0, 36.0)  ≥24 Months to <4 years 68 68 (100.0) (94.7, 100.0) 24 1 (4.2) (0.1, 21.1) ≥4 Years to <10 years 71 71(100.0)  (94.9, 100.0) 19 8 (42.1) (20.3, 66.5)  6 Months afterVaccination 3 ≥24 Months to <10 years 131 108 (82.4) (74.8, 88.5) 46 9(19.6) (9.4, 33.9) ≥24 Months to <4 years 61 49 (80.3) (68.2, 89.4) 24 4(16.7) (4.7, 37.4) ≥4 Years to <10 years 70 59 (84.3) (73.6, 91.9) 22 5(22.7) (7.8, 45.4) PMB2948 (B24) Before Vaccination 1 ≥24 Months to <10years 134 7 (5.2)  (2.1, 10.5) 47 2 (4.3) (0.5, 14.5) ≥24 Months to <4years 67 2 (3.0)  (0.4, 10.4) 26 1 (3.8) (0.1, 19.6) ≥4 Years to <10years 67 5 (7.5)  (2.5, 16.6) 21 1 (4.8) (0.1, 23.8) 1 Month afterVaccination 2 ≥24 Months to <10 years 128 73 (57.0) (48.0, 65.7) 45 4(8.9) (2.5, 21.2) ≥24 Months to <4 years 65 32 (49.2) (36.6, 61.9) 24 2(8.3) (1.0, 27.0) ≥4 Years to <10 years 63 41 (65.1) (52.0, 76.7) 21 2(9.5) (1.2, 30.4) 1 Month after Vaccination 3 ≥24 Months to <10 years126 112 (88.9) (82.1, 93.8) 46 2 (4.3) (0.5, 14.8) ≥24 Months to <4years 63 54 (85.7) (74.6, 93.3) 26 2 (7.7) (0.9, 25.1) ≥4 Years to <10years 63 58 (92.1) (82.4, 97.4) 20 0 (0.0) (0.0, 16.8) 6 Months afterVaccination 3 ≥24 Months to <10 years 129 20 (15.5)  (9.7, 22.9) 47 0(0.0) (0.0, 7.5)  ≥24 Months to <4 years 65 6 (9.2)  (3.5, 19.0) 26 0(0.0) (0.0, 13.2) ≥4 Years to <10 years 64 14 (21.9) (12.5, 34.0) 21 0(0.0) (0.0, 16.1) PMB2707 (B44) Before Vaccination 1 ≥24 Months to <10years 138 0 (0.0) (0.0, 2.6) 50 0 (0.0) (0.0, 7.1)  ≥24 Months to <4years 67 0 (0.0) (0.0, 5.4) 26 0 (0.0) (0.0, 13.2) ≥4 Years to <10 years71 0 (0.0) (0.0, 5.1) 24 0 (0.0) (0.0, 14.2) 1 Month after Vaccination 2≥24 Months to <10 years 130 63 (48.5) (39.6, 57.4) 50 0 (0.0) (0.0,7.1)  ≥24 Months to <4 years 63 36 (57.1) (44.0, 69.5) 26 0 (0.0) (0.0,13.2) ≥4 Years to <10 years 67 27 (40.3) (28.5, 53.0) 24 0 (0.0) (0.0,14.2) 1 Month after Vaccination 3 ≥24 Months to <10 years 134 106 (79.1)(71.2, 85.6) 50 0 (0.0) (0.0, 7.1)  ≥24 Months to <4 years 65 52 (80.0)(68.2, 88.9) 26 0 (0.0) (0.0, 13.2) ≥4 Years to <10 years 69 54 (78.3)(66.7, 87.3) 24 0 (0.0) (0.0, 14.2) 6 Months after Vaccination 3 ≥24Months to <10 years 135 14 (10.4)  (5.8, 16.8) 49 0 (0.0) (0.0, 7.3) ≥24 Months to <4 years 66 8 (12.1)  (5.4, 22.5) 26 0 (0.0) (0.0, 13.2)≥4 Years to <10 years 69 6 (8.7)  (3.3, 18.0) 23 0 (0.0) (0.0, 14.8)Abbreviation: hSBA = serum bactericidal assay using human complement;LLOQ = lower limit of quantitation. Note: LLOQ = 1:16 for A22; 1:8 forA56, B24, and B44. ^(a)N = number of subjects with valid and determinatehSBA titers for the given strain. ^(b)n = Number of subjects withobserved hSBA titer ≥LLOQ for the given strain at the given time point.^(c)Exact 2-sided CI based upon observed proportion of subjects, usingthe Clobber and Pearson method. Program ID: Study B1971017/CPIMM_LLOQ.SAS. Date of Reporting Dataset Creation: 01 JUN. 2017. RuntimeID: 16 JUN. 2017 11:22. File ID: T_2_2_IMM_LLOQ_EVL.HTM.hSBA GMTs. The hSBA GMTs for each of the 4 primary test strains atbaseline, 1 month after the second vaccination, and 1 month after thethird vaccination with bivalent rLP2086 was a secondary endpoint. Table21 provides hSBA GMTs for the 4 primary MnB strains for the evaluableimmunogenicity population.

For Group 1 subjects aged ≥24 months to <4 years and aged ≥4 years to<10 years, hSBA GMTs at baseline were 8.3 and 9.1, respectively, forPMB80 (A22); 4.1 and 5.8, respectively, for PMB2001 (A56); 4.3 and 4.6,respectively, for PMB2948 (B24); and 4.0 in both age strata for PMB2707(B44).

For Group 1 subjects aged ≥24 months to <10 years, hSBA GMTs at baselinewere 8.7 for PMB80 (A22), 4.9 for PMB2001 (A56), 4.5 for PMB2948 (B24),and 4.0 for PMB2707 (B44).

For Group 1 subjects aged ≥24 months to <4 years and aged ≥4 years to<10 years, hSBA GMTs at 1 month after Vaccination 2 were 17.4 and 23.1,respectively, for PMB80 (A22); 103.8 and 90.0, respectively, for PMB2001(A56); 9.1 and 13.7, respectively, for PMB2948 (B24); and 17.1 and 8.2,respectively, for PMB2707 (B44).

For Group 1 subjects aged ≥24 months to <10 years, hSBA GMTs at 1 monthafter Vaccination 2 were 20.1 for PMB80 (A22), 96.6 for PMB2001 (A56);11.1 for PMB2948 (B24), and 11.7 for PMB2707 (B44).

For Group 1 subjects aged ≥24 months to <4 years and aged ≥4 years to<10 years, hSBA GMTs at 1 month after Vaccination 3 were 33.7 and 38.2,respectively, for PMB80 (A22); 175.6 and 191.0, respectively, forPMB2001 (A56); 19.1 and 26.8, respectively, for PMB2948 (B24); and 43.6and 36.5, respectively, for PMB2707 (B44).

For Group 1 subjects aged ≥24 months to <10 years, hSBA GMTs at 1 monthafter Vaccination 3 were 35.8 for PMB80 (A22), 183.3 for PMB2001 (A56);22.6 for PMB2948 (B24), and 39.8 for PMB2707 (B44).

In general, the hSBA GMTs for subjects in Group 2 did not change overtime compared to baseline. For the combined age stratum, hSBA GMTs atany time point ranged from 8.6 to 8.9 for PMB80 (A22); 5.6 to 6.0 forPMB2001 (A56); 4.0 to 4.8 for PMB2948 (B24); and 4.0 at each time pointfor PMB2707 (B44).

TABLE 21 hSBA GMTs for Primary Strains—Evaluable ImmunogenicityPopulation Vaccine Group (as Randomized) Strain (Variant) Group 1 Group2 Sampling Time Point rLP2086 HAV/Saline Age Strata N^(a) GMT^(b) (95%CI)^(c) N^(a) GMT^(b) (95% CI)^(c) PMB80 (A22) Before Vaccination 1 ≥24Months to <10 years 134 8.7 (8.3, 9.1) 47 8.9  (7.8, 10.1) ≥24 Months to<4 years 68 8.3 (7.9, 8.8) 26 8.2 (7.8, 8.7) ≥4 Years to <10 years 669.1 (8.3, 9.9) 21 9.8  (7.2, 13.2) 1 Month after Vaccination 2 ≥24Months to <10 years 130 20.1 (17.4, 23.2) 45 8.6 (7.7, 9.7) ≥24 Monthsto <4 years 64 17.4 (14.2, 21.4) 24 8.0 (NE, NE) ≥4 Years to <10 years66 23.1 (18.9, 28.3) 21 9.4  (7.4, 12.0) 1 Month after Vaccination 3 ≥24Months to <10 years 135 35.8 (30.5, 42.2) 45 8.8 (7.8, 9.8) ≥24 Monthsto <4 years 68 33.7 (26.4, 42.9) 25 8.7  (7.3, 10.3) ≥4 Years to <10years 67 38.2 (30.6, 47.6) 20 8.9  (7.6, 10.4) 6 Months afterVaccination 3 ≥24 Months to <10 years 126 12.4 (10.9, 14.2) 47 8.7 (7.9,9.7) ≥24 Months to <4 years 63 10.9  (9.0, 13.1) 26 8.4 (7.8, 9.1) ≥4Years to <10 years 63 14.2 (11.8, 17.0) 21 9.1  (7.4, 11.3) PMB2001(A56) Before Vaccination 1 ≥24 Months to <10 years 132 4.9 (4.3, 5.5) 475.6 (4.4, 7.2) ≥24 Months to <4 years 67 4.1 (3.9, 4.3) 24 4.9 (3.7,6.6) ≥4 Years to <10 years 65 5.8 (4.6, 7.3) 23 6.5 (4.3, 9.8) 1 Monthafter Vaccination 2 ≥24 Months to <10 years 133 96.6  (83.0, 112.5) 435.8 (4.4, 7.6) ≥24 Months to <4 years 66 103.8  (84.2, 127.9) 21 5.0(3.6, 7.1) ≥4 Years to <10 years 67 90.0  (71.9, 112.7) 22 6.6  (4.2,10.5) 1 Month after Vaccination 3 ≥24 Months to <10 years 139 183.3(156.7, 214.4) 43 6.0 (4.6, 7.7) ≥24 Months to <4 years 68 175.6 (139.1,221.6) 24 4.5 (3.5, 5.7) ≥4 Years to <10 years 71 191.0 (153.9, 237.1)19 8.6  (5.4, 13.8) 6 Months after Vaccination 3 ≥24 Months to <10 years131 31.3 (25.3, 38.7) 46 6.0 (4.6, 7.8) ≥24 Months to <4 years 61 27.0(19.7, 36.9) 24 6.0 (4.0, 8.9) ≥4 Years to <10 years 70 35.7 (26.6,47.8) 22 6.0 (4.2, 8.7) PMB2948 (B24) Before Vaccination 1 ≥24 Months to<10 years 134 4.5 (4.1, 4.9) 47 4.4 (3.9,4.9) ≥24 Months to <4 years 674.3 (3.8, 4.9) 26 4.3 (3.7, 5.1) ≥4 Years to <10 years 67 4.6 (4.0, 5.2)21 4.4 (3.6, 5.4) 1 Month after Vaccination 2 ≥24 Months to <10 years128 11.1  (9.2, 13.5) 45 4.8 (4.0, 5.8) ≥24 Months to <4 years 65 9.1 (7.0, 11.9) 24 4.8 (3.7, 6.2) ≥4 Years to <10 years 63 13.7 (10.3,18.2) 21 4.9 (3.6, 6.6) 1 Month after Vaccination 3 ≥24 Months to <10years 126 22.6 (19.1, 26.8) 46 4.3 (3.9, 4.8) ≥24 Months to <4 years 6319.1 (14.9, 24.5) 26 4.6 (3.8, 5.6) ≥4 Years to <10 years 63 26.8 (21.3,33.9) 20 4.0 (NE, NE) 6 Months after Vaccination 3 ≥24 Months to <10years 129 5.6 (4.8, 6.5) 47 4.0 (NE, NE) ≥24 Months to <4 years 65 5.1(4.1, 6.3) 26 4.0 (NE, NE) ≥4 Years to <10 years 64 6.2 (4.9, 7.7) 214.0 (NE, NE) PMB2707 (B44) Before Vaccination 1 ≥24 Months to <10 years138 4.0 (NE, NE) 50 4.0 (NE, NE) ≥24 Months to <4 years 67 4.0 (NE, NE)26 4.0 (NE, NE) ≥4 Years to <10 years 71 4.0 (NE, NE) 24 4.0 (NE, NE) 1Month after Vaccination 2 ≥24 Months to <10 years 130 11.7  (9.3, 14.7)50 4.0 (NE, NE) ≥24 Months to <4 years 63 17.1 (11.8, 24.8) 26 4.0 (NE,NE) ≥4 Years to <10 years 67 8.2  (6.3, 10.6) 24 4.0 (NE, NE) 1 Monthafter Vaccination 3 ≥24 Months to <10 years 134 39.8 (30.6, 51.6) 50 4.0(NE, NE) ≥24 Months to <4 years 65 43.6 (29.9, 63.6) 26 4.0 (NE, NE) ≥4Years to <10 years 69 36.5 (25.2, 52.7) 24 4.0 (NE, NE) 6 Months afterVaccination 3 ≥24 Months to <10 years 135 5.1 (4.4, 5.9) 49 4.0 (NE, NE)≥24 Months to <4 years 66 5.2 (4.2, 6.4) 26 4.0 (NE, NE) ≥4 Years to <10years 69 5.0 (4.1, 6.2) 23 4.0 (NE, NE) Abbreviations: GMT = geometricmean titer; hSBA = serum bactericidal assay using human complement; LLOQ= lower limit of quantitation; NE = not estimable. Note: LLOQ = 1:16 forA22; 1:8 for A56, B24, and B44. Titers below the LLOQ were set to 0.5 ×LLOQ for analysis. ^(a)N = number of subjects with valid and determinatehSBA titers for the given strain. ^(b)GMTs were calculated using allsubjects with valid and determinate hSBA titers at the given time point.^(c)CIs are back transformations of confidence levels based on theStudent t distribution for the mean logarithm of the hSBA titers.

Subgroup analyses of hSBA GMTs for each of the 4 primary MnB teststrains are presented for the evaluable immunogenicity population bysex, race, and country, and for the mITT population. There were noclinically important differences observed in the subgroup analysesperformed.

Immunopersistence: hSBA GMTs

The hSBA GMTs for each of the 4 primary test strains at 6 months afterthe third vaccination with bivalent rLP2086 was a secondary endpoint.Table 21 provides hSBA GMTs for the 4 primary MnB strains for theevaluable immunogenicity population. Overall, there was a decreaseobserved from 1 month after the third vaccination to 6 months after thethird vaccination in hSBA GMTs for each of the 4 primary test strainsfor Group 1 subjects in both age strata.

For Group 1 subjects aged ≥24 months to <4 years, from 1 month after thethird vaccination to 6 months after the third vaccination, hSBA GMTsdecreased from 33.7 to 10.9 for PMB80 (A22), 175.6 to 27.0 forPMB2001(A56), 19.1 to 5.1 for PMB2948 (B24), and 43.6 to 5.2 for PMB2707(B44).

For Group 1 subjects aged ≥4 years to <10 years, from 1 month after thethird vaccination to 6 months after the third vaccination, hSBA GMTsdecreased from 38.2 to 14.2 for PMB80 (A22), 191.0 to 35.7 for PMB2001(A56), 26.8 to 6.2 for PMB2948 (B24), and 36.5 to 5.0 for PMB2707 (B44).

For Group 1 subjects aged ≥24 months to <10 years, from 1 month afterthe third vaccination to 6 months after the third vaccination, hSBA GMTsdecreased from 35.8 to 12.4 for PMB80 (A22), 183.3 to 31.3 forPMB2001(A56), 22.6 to 5.6 for PMB2948 (B24), and 39.8 to 5.1 for PMB2707(B44).

In general, the hSBA GMTs for subjects in Group 2 did not change overtime compared to baseline.

Defined hSBA Titers

The proportions of subjects aged ≥24 months to <4 years, ≥4 years to <10years, and in the combined age stratum, achieving hSBA titers of ≥1:4,≥1:8, ≥1:16, ≥1:32, ≥1:64, and ≥1:128 for each of the 4 primary teststrains at baseline, 1 month after the second vaccination, and 1 monthafter the third vaccination with bivalent rLP2086 was a secondaryimmunogenicity endpoint.

The proportion of subjects achieving defined hSBA titers for the 4primary MnB strains was assessed for the evaluable immunogenicitypopulation.

Subjects who achieved an hSBA titer ≥1:4 and ≥1:16 are described below.An hSBA titer of ≥1:4 is widely recognized as the correlate ofprotection against IMD; however, a more conservative hSBA titer of ≥1:16has been considered a level indicative of a 4-fold vaccine effect forsubjects seronegative before vaccination.

The proportion of subjects aged ≥24 months to <4 years, and ≥4 years to<10 years, in Group 1 with an hSBA titer ≥1:4 at baseline was 5.9% and19.7%, respectively, for PMB80 (A22); 3.0% and 18.5%, respectively, forPMB2001 (A56); 4.5% and 9.0%, respectively, for PMB2948 (B24); and 0.0%and 1.4%, respectively for PMB2707 (B44). Subjects aged ≥24 months to <4years, and ≥4 years to <10 years, in Group 1 with an hSBA titer ≥1:16 atbaseline was 4.4% and 13.6%, respectively, for PMB80 (A22); 1.5% and15.4%, respectively, for PMB2001 (A56); 3.0% and 6.0%, respectively, forPMB2948 (B24); and 0.0% for both age strata for PMB2707 (B44).

The proportion of Group 1 subjects in the combined age stratum with anhSBA titer □1:4 and □1:16 at baseline was 12.7% and 9.0%, respectively,for PMB80 (A22); 10.6% and 8.3%, respectively, for PMB2001 (A56); 6.7%and 4.5%, respectively, for PMB2948 (B24); and 0.7% and 0.0%,respectively, for PMB2707 (B44).

The proportion of subjects aged ≥24 months to <4 years, and ≥4 years to<10 years, in Group 1 with an hSBA titer ≥1:4 at 1 month after thesecond vaccination was 65.6% and 83.3%, respectively, for PMB80 (A22);100.0% for both age strata for PMB2001 (A56); 53.8% and 68.3%,respectively, for PMB2948 (B24); and 49.3% and 66.7%, respectively, forPMB2707 (B44). Subjects aged ≥24 months to <4 years, and ≥4 years to <10years, in Group 1 with an hSBA titer ≥1:16 at 1 month after the secondvaccination was 59.4% and 78.8%, respectively, for PMB80 (A22); 98.5%and 100.0%, respectively, for PMB2001 (A56); 43.1% and 58.7%,respectively, for PMB2948 (B24); and 31.3% and 55.6%, respectively, forPMB2707 (B44).

The proportion of Group 1 subjects in the combined age stratum with anhSBA titer ≥1:4 and ≥1:16 at 1 month after the second vaccination was74.6% and 69.2%, respectively, for PMB80 (A22); 100.0% and 99.2%,respectively, for PMB2001 (A56); 60.9% and 50.8%, respectively, forPMB2948 (B24); and 57.7% and 43.1%, respectively, for PMB2707 (B44).

The proportion of subjects aged ≥24 months to <4 years, and ≥4 years to<10 years, in Group 1 with an hSBA titer ≥1:4 at 1 month after the thirdvaccination was 86.8% and 98.5%, respectively, for PMB80 (A22); 100.0%for each age strata for PMB2001 (A56); 90.5% and 95.2% for PMB2948(B24); and 81.5% and 82.6%, respectively for PMB2707 (B44). Subjectsaged ≥24 months to <4 years, and ≥4 years to <10 years, in Group 1 withan hSBA titer ≥1:16 at 1 month after the third vaccination was 83.8% and91.0%, respectively, for PMB80 (A22); 100.0% for each age stratum forPMB2001 (A56); 81.0% and 88.9%, respectively, for PMB2948 (B24); and81.5% to 82.6% and 80.0% and 75.4%, respectively, for PMB2707 (B44).

The proportion of Group 1 subjects in the combined age stratum with anhSBA titer ≥1:4 and ≥1:16 at 1 month after the third vaccination was92.6% and 87.4%, respectively, for PMB80 (A22); 100.0% and 100.0%,respectively, for PMB2001 (A56); 92.9% and 84.9%, respectively, forPMB2948 (B24); and 82.1% and 77.6%, respectively, for PMB2707 (B44).

In general, the proportion of Group 2 subjects achieving defined hSBAtiters did not change overtime compared to baseline.

Results for the mITT population were similar to those of the evaluableimmunogenicity population.

Immunopersistence: Defined hSBA Titers

The proportions of subjects aged ≥24 months to <4 years, ≥4 years to <10years, and in the combined age stratum, achieving hSBA titers of ≥1:4,21:8, ≥1:16, ≥1:32, ≥1:64, and 1:128 for each of the 4 primary teststrains at 6 months after the third vaccination with bivalent rLP2086was a secondary immunogenicity endpoint. The proportion of subjectsachieving defined hSBA titers for the 4 primary MnB strains was assessedfor the evaluable immunogenicity population.

Overall, there was a decrease observed in the proportion of Group 1subjects in both age strata who achieved defined hSBA titers from 1month after the third vaccination to 6 months after the thirdvaccination.

For Group 1 subjects aged ≥24 months to <4 years and aged ≥4 years to<10 years, from 1 month after the third vaccination to 6 months afterthe third vaccination, the proportion of subjects with an hSBA titer≥1:4 decreased from 86.8% to 25.4% and 98.5% to 55.6%, respectively, forPMB80 (A22); 100.0% to 82.0% and 100.0% to 85.7%, respectively forPMB2001 (A56); 90.5% to 13.8% and 95.2% to 26.6%, respectively, forPMB2948 (B24); and 81.5% to 13.6% and 82.6% to 13.0%, respectively, forPMB2707 (B44).

For Group 1 subjects in the combined age stratum, from 1 month after thethird vaccination to 6 months after the third vaccination, theproportion of subjects with an hSBA titer ≥1:4 decreased from 92.6% to40.5% for PMB80 (A22); 100.0% to 84.0% for PMB2001 (A56); 92.9% to 20.2%for PMB2948 (B24); and 82.1% to 13.3% for PMB2707 (B44).

For Group 1 subjects aged ≥24 months to <4 years and aged ≥4 years to<10 years, from 1 month after the third vaccination to 6 months afterthe third vaccination, the proportion of subjects with an hSBA titer≥1:16 decreased from 83.8% to 19.0% and 91.0% to 46.0%, respectively,for PMB80 (A22); 100.0% to 77.0% and 100.0% to 82.9%, respectively, forPMB2001 (A56); 81.0% to 9.2% and 88.9% to 20.3%, respectively, forPMB2948 (B24); and 80.0% to 9.1% and 75.4% 7.2%, respectively, forPMB2707 (B44). For Group 1 subjects in the combined age stratum, from 1month after the third vaccination to 6 months after the thirdvaccination, the proportion of subjects with an hSBA titer ≥1:16decreased from 87.4% to 32.5% for PMB80 (A22); 100.0% to 80.2% forPMB2001 (A56); 84.9% to 14.7% for PMB2948 (B24); and 77.6% to 8.1% forPMB2707 (B44).

In general, the proportion of Group 2 subjects achieving defined hSBAtiters did not change overtime compared to baseline.

Exploratory Immunogenicity Endpoints

The analysis of some exploratory immunogenicity endpoints was based onhSBA results for strains PMB2001 (A56) and PMB2707 (B44) for half of thesubjects, and strains PMB80 (A22) and PMB2948 (B24) for the remaininghalf. The exploratory endpoint analyzed was hSBA titer with a ≥4-foldincrease from baseline.

hSBA Titer 4-Fold Increase From Baseline Table 22 presents theproportion of subjects with hSBA titers with a ≥4-fold rise frombaseline for the 4 primary test strains.

The proportion of Group 1 subjects aged ≥24 months to <4 years and aged≥4 years to <10 years achieving a ≥4-fold rise in hSBA titer frombaseline to 1 month after Vaccination 2 was 56.3% and 63.6%,respectively for PMB80 (A22); 100.0% and 82.1%, respectively for PMB2001(A56); 43.1% and 54.0%, respectively, for PMB2948 (B24); and 54.0% and31.3%, respectively, for PMB2707 (B44).

The proportion of Group 1 subjects in the combined age stratum achievinga ≥4-fold rise in hSBA titer from baseline to 1 month after Vaccination2 was 60.0% for PMB80 (A22), 91.0% for PMB2001 (A56), 48.4% for PMB2948(B24), and 42.3% for PMB2707 (B44).

The proportion of Group 1 subjects aged ≥24 months to <4 years and aged≥4 years to <10 years, achieving a ≥4-fold rise in hSBA titer frombaseline to 1 month after Vaccination 3 was 79.4% and 77.6%,respectively for PMB80 (A22); 98.5% and 88.7%, respectively for PMB2001(A56); 77.8% and 82.5%, respectively, for PMB2948 (B24); and 78.5% and75.4%, respectively, for PMB2707 (B44).

The proportion of Group 1 subjects in the combined age stratum achievinga ≥4-fold rise in hSBA titer from baseline to 1 month after Vaccination3 was 78.5% for PMB80 (A22), 93.5% for PMB2001 (A56), 80.2% for PMB2948(B24), and 76.9% for PMB2707 (B44).

The proportion of Group 1 subjects aged ≥24 months to <4 years and aged≥4 years to <10 years achieving a ≥4-fold rise in hSBA titer frombaseline to 6 months after Vaccination 3 was 19.0% and 36.5%,respectively for PMB80 (A22); 75.4% and 64.3%, respectively for PMB2001(A56); 6.2% and 17.2%, respectively, for PMB2948 (B24); and 7.6% and7.2%, respectively, for PMB2707 (B44).

The proportion of Group 1 subjects in the combined age stratum achievinga ≥4-fold rise in hSBA titer from baseline to 6 months after Vaccination3 was 27.8% for PMB80 (A22), 69.5% for PMB2001 (A56), 11.6% for PMB2948(B24), and 7.4% for PMB2707 (B44). Similar results were observed for themITT population.

TABLE 22 Subjects With hSBA Titer 4-Fold Rise for Primary Strains—Evaluable Immunogenicity Population Vaccine Group (as Randomized) Strain(Variant) Group 1 Group 2 Sampling Time Point rLP2086 HAV/Saline AgeStrata N^(a) n^(b) (%) (95% CI)^(c) N^(a) n^(b) (%) (95% CI)^(c) hSBAtiter fold rise ≥4 from baseline^(d) PMB80 (A22) 1 Month afterVaccination 2 ≥24 Months to <10 years 130 78 (60.0) (51.0, 68.5) 45 0(0.0) (0.0, 7.9) ≥24 Months to <4 years 64 36 (56.3) (43.3, 68.6) 24 0(0.0)  (0.0, 14.2) ≥4 Years to <10 years 66 42 (63.6) (50.9, 75.1) 21 0(0.0)  (0.0, 16.1) 1 Month after Vaccination 3 ≥24 Months to <10 years135 106 (78.5) (70.6, 85.1) 45 1 (2.2)  (0.1, 11.8) ≥24 Months to <4years 68 54 (79.4) (67.9, 88.3) 25 1 (4.0)  (0.1, 20.4) ≥4 Years to <10years 67 52 (77.6) (65.8, 86.9) 20 0 (0.0)  (0.0, 16.8) 6 Months afterVaccination 3 ≥24 Months to <10 years 126 35 (27.8) (20.2, 36.5) 47 2(4.3)  (0.5, 14.5) ≥24 Months to <4 years 63 12 (19.0) (10.2, 30.9) 26 2(7.7)  (0.9, 25.1) ≥4 Years to <10 years 63 23 (36.5) (24.7, 49.6) 21 0(0.0)  (0.0, 16.1) PMB2001 (A56) 1 Month after Vaccination 2 ≥24 Monthsto <10 years 133 121 (91.0) (84.8, 95.3) 43 4 (9.3)  (2.6, 22.1) ≥24Months to <4 years 66 66 (100.0)  (94.6, 100.0) 21 1 (4.8)  (0.1, 23.8)≥4 Years to <10 years 67 55 (82.1) (70.8, 90.4) 22 3 (13.6)  (2.9, 34.9)1 Month after Vaccination 3 ≥24 Months to <10 years 139 130 (93.5)(88.1, 97.0) 43 8 (18.6)  (8.4, 33.4) ≥24 Months to <4 years 68 67(98.5)  (92.1, 100.0) 24 1 (4.2)  (0.1, 21.1) ≥4 Years to <10 years 7163 (88.7) (79.0, 95.0) 19 7 (36.8) (16.3, 61.6) 6 Months afterVaccination 3 ≥24 Months to <10 years 131 91 (69.5) (60.8, 77.2) 46 6(13.0)  (4.9, 26.3) ≥24 Months to <4 years 61 46 (75.4) (62.7, 85.5) 243 (12.5)  (2.7, 32.4) ≥4 Years to <10 years 70 45 (64.3) (51.9, 75.4) 223 (13.6)  (2.9, 34.9) PMB2948 (B24) 1 Month after Vaccination 2 ≥24Months to <10 years 128 62 (48.4) (39.5, 57.4) 45 3 (6.7)  (1.4, 18.3)≥24 Months to <4 years 65 28 (43.1) (30.8, 56.0) 24 1 (4.2)  (0.1, 21.1)≥4 Years to <10 years 63 34 (54.0) (40.9, 66.6) 21 2 (9.5)  (1.2, 30.4)1 Month after Vaccination 3 ≥24 Months to <10 years 126 101 (80.2)(72.1, 86.7) 46 2 (4.3)  (0.5, 14.8) ≥24 Months to <4 years 63 49 (77.8)(65.5, 87.3) 26 2 (7.7)  (0.9, 25.1) ≥4 Years to <10 years 63 52 (82.5)(70.9, 90.9) 20 0 (0.0)  (0.0, 16.8) 6 Months after Vaccination 3 ≥24Months to <10 years 129 15 (11.6) (6.7, 18.5) 47 0 (0.0) (0.0, 7.5) ≥24Months to <4 years 65 4 (6.2) (1.7, 15.0) 26 0 (0.0)  (0.0, 13.2) ≥4Years to <10 years 64 11 (17.2) (8.9, 28.7) 21 0 (0.0)  (0.0, 16.1)PMB2707 (B44) 1 Month after Vaccination 2 ≥24 Months to <10 years 130 55(42.3) (33.7, 51.3) 50 0 (0.0) (0.0, 7.1) ≥24 Months to <4 years 63 34(54.0) (40.9, 66.6) 26 0 (0.0) (0.0, 13.2) ≥4 Years to <10 years 67 21(31.3) (20.6, 43.8) 24 0 (0.0) (0.0, 14.2) 1 Month after Vaccination 3≥24 Months to <10 years 134 103 (76.9) (68.8, 83.7) 50 0 (0.0) (0.0,7.1) ≥24 Months to <4 years 65 51 (78.5) (66.5, 87.7) 26 0 (0.0)  (0.0,13.2) ≥4 Years to <10 years 69 52 (75.4) (63.5, 84.9) 24 0 (0.0)(0.0,14.2) 6 Months after Vaccination 3 ≥24 Months to <10 years 135 10(7.4) (3.6, 13.2) 49 0 (0.0) (0.0, 7.3) ≥24 Months to <4 years 66 5(7.6) (2.5, 16.8) 26 0 (0.0)  (0.0, 13.2) ≥4 Years to <10 years 69 5(7.2) (2.4, 16.1) 23 0 (0.0)  (0.0, 14.8) Abbreviations: hSBA = serumbactericidal assay using human complement; LLOQ = lower limit ofquantitation; LOD = limit of detection. Note: LLOQ = 1:16 for A22; 1:8for A56, B24, and B44. Note: The 4-fold increase is defined as follows:(1) For subjects with a baseline hSBA titer below the LOD (hSBA titer<1:4), a response is defined as an hSBA titer ≥1:16 or the LLOQ(whichever titer is higher). (2) For subjects with a baseline hSBA titer≥ LOD and <LLOQ, a response is defined as an hSBA titer ≥4 times theLLOQ. (3) For subjects with a baseline hSBA titer ≥LLOQ, a response isdefined as an hSBA titer ≥4 times the baseline titer. ^(a)For hSBA titerfold rise ≥4 from baseline, N = number of subjects with valid anddeterminate hSBA titers for the given strain at both the specified timepoint and baseline. ^(b)For hSBA titer fold rise ≥4 from baseline, n =number of subjects who achieved hSBA titer fold rise .el from baselinefor the given strain. ^(c)Exact 2-sided CI based upon observedproportion of subjects, using the Clobber and Pearson method.^(d)Baseline is defined as the blood draw prior to Vaccination 1.

Additional Immunogenicity Analyses

Assessment of Missing hSBA Data for Primary MnB Test Strains

Only valid and determinate hSBA results were included in allimmunogenicity analyses. The hSBA results were excluded from theimmunogenicity analysis (or considered to be missing) for the followingreasons:

-   -   The subject withdrew from the study.    -   The subject did not have blood samples for testing but was not        withdrawn from the study.    -   The quantity of blood was insufficient to perform the assay.        This was entered as “quantity not sufficient” for the assay        results.    -   The sample was tested but a numerical titer could not be        reliably determined. This was entered as “indeterminate” for the        assay results.

Reverse Cumulative Distribution Curves

The RCDCs of the proportions of subjects exhibiting an hSBA response(LLOQ) for each of the 4 primary strains and at each sampling timepoint, for the combined age stratum were assessed. RCDCs for each of the4 primary strains and at each sampling time point, for subjects aged ≥24months to <4 years were also assessed. RCDCs for each of the 4 primarystrains and at each sampling time point, for subjects aged 4 years to<10 years were assessed.

The RCDCs showed the majority of subjects in both age strata exhibited ameasurable hSBA response to each of the primary MnB test strains at 1month after the second and third dose of bivalent rLP2086.

Immunogenicity Conclusions

The primary objectives of this study were to describe subject immuneresponse to bivalent rLP2086 as measured by hSBA against 4 primary MnBtest strains, 2 expressing an LP2086 subfamily A protein and 2expressing an LP2086 subfamily B protein, measured 1 month after thethird vaccination in healthy subjects aged ≥24 months to <4 years and ≥4years to <10 years. The description of immune responses for the combinedage stratum (≥24 months to <10 years) was a secondary objective. Theendpoints for the primary objectives were the proportions of subjects ineach age stratum achieving hSBA titers ≥LLOQ for each of the 4 primaryMnB strains 1 month after the third vaccination.

A robust immune response was observed for children aged ≥24 months to<10 years 1 month after the third dose of bivalent rLP2086, as confirmedby the proportion of subjects achieving an hSBA titer ≥LLOQ (1:8 forA56, B24 and B44; 1:16 for A22) for each of the 4 primary MnB teststrains ranging from 80.0% to 100.0% for subjects aged ≥24 months to <4years and from 78.3% to 100.0% for subjects ≥4 years to <10 years after3 doses. The proportion of subjects in the combined age stratum with anhSBA titer ≥LLOQ for each of the 4 primary MnB test strains 1 monthafter the third vaccination ranged from 79.1% to 100.0%. These findingsare further supported by substantial GMTs (range 19.1 to 191) and in theproportion of subjects achieving an hSBA titer ≥1:4 (81.5% to 100%) or≥1:16 (75.4% to 100%) against each of the 4 primary MnB test strainsafter 3 doses of bivalent rLP2086 compared to baseline across both agestrata. Additionally, the proportion of subjects in the combined agestratum achieving an hSBA fold rise ≥4 from baseline to 1 month afterthe third vaccination for each of the 4 primary MnB test strains rangedfrom 76.9% to 93.5%.

The secondary objective of the study was to describe immune responses 1month after the second dose of bivalent rLP2086, as assessed by ≥LLOQresponses, defined hSBA titers and hSBA GMTs for the 2 age strata andthe combined age stratum. For the combined age stratum, the proportionof subjects achieving an hSBA titer ≥LLOQ ranged from 48.5% to 100.0%with no meaningful differences observed between the younger and olderage strata. These findings are further supported by the combined agestratum with increases in GMTs (range 11.1 to 96.6) and in theproportion of subjects achieving an hSBA titer ≥1:4 (57.7% to 100%) or≥1:16 (43.1% to 100%) after 2 doses of bivalent rLP2086 compared tobaseline against each of the 4 primary MnB test strains. GMTs weresimilar between the 2 age strata. Additionally, the proportion ofsubjects in the combined age stratum achieving an hSBA fold rise ≥4 frombaseline to 1 month after the second vaccination for each of the 4primary MnB test strains ranged from 42.3% to 91.0%.

Immunopersistence was also assessed at 6 months after the third dose ofbivalent rLP2086 with the proportion of subjects with an hSBA titer≥LLOQ declining from 79.1% to 100% 1 month after Vaccination 3 to 10.4%to 82.4% at 6 months after the third vaccination for the combined agestratum. No differences between the 2 age strata were observed exceptfor A22, for which older children had a higher proportion of subjectsachieving a titer ≥LLOQ than the younger children (46%, 95% C1 33.4,59.1 vs 19%, 95% CI 10.2, 30.9). However, baseline prevaccination ratesof titers ≥LLOQ were greater for A22 in the older age stratum (13.6% vs4.4%). A similar trend was also observed for the combined age stratumfor the proportion of subjects with a protective hSBA titer ≥1:4,ranging from 13.3% to 84.0% and GMTs, ranging from 5.1 to 31.3 at 6months after the third vaccination.

In summary, bivalent rLP2086 given as 3 doses on a 0-, 2-, and 6-monthschedule elicits a robust immune response among toddlers and childrenaged ≥24 months to <10 years with protective antibody titers achieved asmeasured by hSBA in a high proportion of subjects after the third dose.No clinically meaningful differences were observed between toddlers aged≥24 months to <4 years and children aged ≥4 years to <10 years. Antibodyresponses decline 6 months after the third dose, but remain higher thanprevaccination baseline rates.

Discussion and Overall Conclusions

Immunogenicity Discussion

Immunogenicity results from this Phase 2 study of a 3-dose regimen (0-,2-, and 6-month schedule) of bivalent rLP2086 given to toddlers andchildren aged ≥24 months to <10 years are consistent with previousstudies in adolescents and young adults.

Immunogenicity responses to bivalent rLP2086 vaccination were measuredin validated hSBAs using 4 primary MnB test strains, each expressingfHBP variants heterologous to the vaccine component antigens, usingcriteria more stringent than the accepted correlate of protection (hSBAtiter ≥1:4). Based on an hSBA titer ≥LLOQ for the 4 primary MnB teststrains 1 month after Vaccination 3, the toddlers and childrenparticipating in this study had similar immune responses compared toadolescents (10 years to <19 years) participating in Study B1971009,with proportions of subjects achieving an hSBA titer ≥LLOQ after thethird vaccination (0-, 2-, 6-month schedule) ranging from 79.1% to 100%in this study and 87.1% to 99.5% in Study B1971009. Meaningfuldifferences in the proportion of subjects with an hSBA titer ≥LLOQ forthe 4 primary test strains 1 month after Vaccination 3 between these 2studies are not apparent, despite the fact that Study B1971009 had amuch higher proportion of adolescent subjects with a prevaccination hSBAtiter ≥LLOQ compared to the toddlers and children in this study,particularly for the A22 (33.2% vs 9%, respectively) and A56 (27.5% vs8.3%, respectively) test strains. Bivalent rLP2086 appears to be highlyimmunogenic in the ≥24 months to <10 years age population and is likelyto offer protection against MnB infection similarly to that expected foradolescents based on the hSBA correlate of protection.

With regard to the secondary objectives, immune responses in this studyfor the combined age stratum (≥24 months to <10 years) 1 month after thesecond dose of bivalent rLP2086 were relatively robust for the 4 primaryMnB test strains, with the proportion of subjects achieving an hSBAtiter ≥LLOQ ranging from 48.5% to 100%. With exception of strain A56,these responses were lower than immune responses observed amongadolescents (10 years to <19 years) participating in Study B1971009receiving 2 doses of bivalent rLP2086 given 2 months apart, ranging from64% to 99.1%. Immunopersistence was assessed in this study for toddlersand children by measuring hSBA titers 6 months after Vaccination 3. Theproportion of subjects achieving an hSBA titer ≥LLOQ declined from arange of 79.1% to 100% 1 month after Vaccination 3 to a range of 10.4%to 82.4% 6 months after Vaccination 3. GMTs and the proportion ofsubjects with defined hSBA titers also declined 6 months afterVaccination 3. Although the proportions of subjects achieving an hSBAtiter ≥LLOQ 6 months after Vaccination 3 in this study (10.4% to 82.4%)were lower than for adolescents (11 years to <19 years) participating inStudy 6108A1-2001 (36.7% to 89.4%), they are still higher than theproportion of subjects aged ≥24 months to <10 years with an hSBA titer≥LLOQ at baseline (0% to 9%). It is well established that meningococcalcolonization rates increase with age through early adulthood.Differences between subjects aged ≥24 months to <10 years and older agegroups may be partially attributable to the proportion of subjects witha baseline titer ≥LLOQ, which were as high as 27.5% (A56) and 33.2%(A22) in Study B1971009, and 7.4% to 13.4% for subfamily A strains inStudy 6108A1-2001. Additionally, a recent study showed that carriage ofdisease-associated serogroup B strains was higher in subjects withprotective hSBA titers (before vaccination), and that vaccination didnot impact subsequent carriage of those disease-associated strains. Thissuggests that carriage may impact baseline hSBA titers and thatpersistence or recolonization after vaccination may be contributing to agreater proportion of subjects with an hSBA titer ≥LLOQ observed amongadolescents 6 months after vaccination compared to the toddlers andchildren in this study, who we speculate are less likely to be colonizedand have lower percentages of hSBA titers ≥LLOQ at baseline.Immunopersistence studies with monovalent conjugate meningococcalserogroup C vaccine in infants and children similarly show thatprotective immunity rapidly wanes. However, even among a cohort ofchildren with percentages of hSBA titers >1:8 for serogroup C at only46.9% 4 years after completion of the primary dose, a subsequent boosterdose provided titers >1:8 in 100% of subjects 1 month and 1 year afterthe booster. This indicates that even those considered seronegativeprior to the booster dose have a strong anamnestic response whichpersists for up to 1 year after booster vaccination. Postboosterresponse and persistence studies are therefore warranted amongindividuals who received their primary series of bivalent rLP2086 astoddlers and children to provide further insights into the utility of abooster dose in providing protection against IMD through adolescence andearly adulthood.

In summary, bivalent rLP2086 administered on a 0-, 2-, and 6-monthschedule is highly immunogenic among toddlers and children aged ≥24months to <10 years with protective immune responses achieved asmeasured by hSBA in a high proportion of subjects after the third dose.Immune responses, as measured in this study, appear to be similar tothat observed in prior studies among adolescents 1 month after thesecond and third doses. The 3-dose regimen appears to provide high ratesof protective immunity in toddlers and children aged ≥24 months to <10years.

Overall Conclusions

In conclusion, bivalent rLP2086 administered to toddlers and childrenaged ≥24 months to <10 years in a 3-dose series on a 0-, 2-, and 6-monthschedule elicits a robust immune response by the majority of subjectsafter the second and third doses, with protective antibody titersachieved after the third dose as measured by hSBAs. hSBA titersdecreased 6 months after a 3-dose series. The vaccine, as administeredin this study, was safe and well tolerated with an acceptable safetyprofile for toddlers and children aged ≥24 months to <10 years.

EXAMPLE 20: A Phase 2, Randomized, Controlled, Observer-Blinded StudyConducted to Describe the Immunogenicity, Safety, and Tolerability of aNeisseria meningitidis Serogroup B Bivalent Recombinant Lipoprotein 2086Vaccine (Bivalent rLP2086) When Administered to Healthy Toddlers Aged 12to <18 Months or 18 to <24 Months (B1971035-syn)

Objectives

Primary Immunogenicity Objectives:

-   -   To describe the immune response as measured by serum        bactericidal assay using human complement (hSBA) performed with        4 primary Neisseria meningitidis serogroup B (MnB) strains, 2        expressing an LP2086 subfamily A protein and 2 expressing an        LP2086 subfamily B protein, measured 1 month after the third        vaccination with bivalent rLP2086, in healthy toddlers aged 12        to <18 months at study entry.    -   To describe the immune response as measured by hSBA performed        with 4 primary MnB strains, 2 expressing an LP2086 subfamily A        protein and 2 expressing an LP2086 subfamily B protein, measured        1 month after the third vaccination with bivalent rLP2086, in        healthy toddlers aged 18 to <24 months at study entry.

Primary Safety Objective:

-   -   To evaluate the safety profile of bivalent rLP2086 compared to a        control (hepatitis A virus [HAV] vaccine), as measured by local        reactions, systemic events, adverse events (AEs), serious        adverse events (SAEs), newly diagnosed chronic medical        conditions (NDCMCs), medically attended events (MAEs), and        immediate AEs in healthy toddlers 12 to <18 months and 18 to <24        months of age at study entry, and in both age strata combined.

Secondary Immunogenicity Objectives:

-   -   To describe the immune response as measured by hSBA performed        with 4 primary MnB test strains, 2 expressing an LP2086        subfamily A protein and 2 expressing an LP2086 subfamily B        protein, measured 1 month after the third vaccination with        bivalent rLP2086, in healthy toddlers aged 12 to <24 months at        study entry (ie, both age strata combined).    -   To describe the immune response as measured by hSBA performed        with 4 primary MnB test strains, 2 expressing an LP2086        subfamily A protein and 2 expressing an LP2086 subfamily B        protein, measured 1 month after the second vaccination and 6,        12, 24, 36, and 48 months after the third vaccination in healthy        toddlers aged 12 to <18 months and 18 to <24 months at study        entry, and in both age strata combined.

Exploratory Immunogenicity Objectives:

-   -   To further describe the immune response as measured by hSBA        performed with 4 primary MnB test strains, 2 expressing an        LP2086 subfamily A protein and 2 expressing an LP2086 subfamily        B protein, measured 1 month after the second vaccination and 1,        6, 12, 24, 36, and 48 months after the third vaccination with        bivalent rLP2086 in healthy toddlers aged 12 to <18 months and        18 to <24 months at study entry, and in both age strata        combined.    -   To further describe the immune response as measured by hSBA to        secondary MnB test strains expressing LP2086 subfamily A and B        proteins, at 1 month after the second vaccination and 1, 6, 12,        24, 36, and 48 months after the third vaccination in healthy        toddlers aged 12 to <18 months and 18 to <24 months at study        entry, and in both age strata combined.

Methods

Study Design:

The study was a Phase 2, randomized, active-controlled,observer-blinded, sponsor-unblinded, multicenter study in whichapproximately 396 healthy toddlers stratified by age, 12 to <18 monthsor 18 to <24 months old, were randomly assigned in a 2:1 ratio toreceive bivalent rLP2086 (either of 2 dose levels [60 μg or 120 μg]) ora licensed pediatric HAV vaccine (0.5 mL)/sterile saline solution forinjection (0.5-mL of 0.85% sodium chloride).

The study was conducted in 2 stages. Stage 1 assessed vaccineimmunogenicity, safety, and tolerability across 2 phases: asentinel-enrollment phase and an expanded-enrollment phase. Stage 2assessed the duration of the immune response to bivalent rLP2086.

For immunogenicity, all data through 1 month after Vaccination 3 (Visit7) are presented with the exception of secondary MnB test strain datasupporting an exploratory objective.

The final report includes all immunogenicity data through the completionof Stage 2 and safety data for the period after Visit 8 until the end ofthe study (Visit 13, 48 months after Vaccination 3).

The Stage 1 sentinel-enrollment phase was planned to include a total of4 sentinel cohorts: 2 age strata for each dose level (60 μg or 120 μg)of bivalent rLP2086. The younger-aged sentinel cohorts were composed ofsubjects aged 12 to <15 months and the older-aged sentinel cohorts werecomposed of subjects aged 18 to <24 months. Each of the 4 sentinelcohorts was planned to enroll approximately 33 subjects. Thesentinel-enrollment phase was staggered with reviews by an IRC atpre-specified points and stopping rules applied. The 120-μg dose levelsentinel cohorts did not proceed until the 60-μg dose level wasevaluated by the IRC as safe and tolerable in the sentinel cohort of thesame age. The younger-aged 120-μg dose level sentinel cohort did notproceed until this dose level was evaluated by the IRC as safe andtolerable in the older-aged 120-μg dose level sentinel cohort.

Prior to the Stage 1 expanded-enrollment phase, the IRC reviewed allpost-Vaccination 1, 7-day e-diary and SAE data obtained from sentinelsubjects. Based on the review, the IRC selected the 120-μg bivalentrLP2086 dose level to be studied in the Stage 1 expanded-enrollmentphase for both age strata. The younger-aged expanded-enrollment cohort(enrolling an additional 132 subjects) was extended to subjects aged 12to <18 months and stratified by age into 2 subsets: aged 12 to <15months and aged 15 to <18 months. The older-aged expanded enrollmentcohort (enrolling an additional 132 subjects) enrolled subjects aged 18to <24 months during the expanded-enrollment phase. The total studyduration for subjects completing only Stage 1 will be approximately 18months. The visit schedule for Stage 1 is presented in Table 23.

Stage 2 includes only those subjects randomly assigned to bivalentrLP2086 (irrespective of dose level). The total study duration forsubjects who complete Stage 2 will be approximately 4.5 years (54months). The visit schedule for Stage 2 is presented in Table 24.

Bivalent rLP2086 was administered at Months 0, 2, and 6 (Visits 1, 4,and 6). Pediatric HAV vaccine was administered at Months 0 and 6 (Visits1 and 6), and saline was administered at Month 2 (Visit 4) to maintainthe study blind.

TABLE 23 Stage 1 Visit Schedule Visit Identifier 1 2 3 4 5 6 7 8 9^(a)10^(a) Time Period Month Week Month Month Month Month Month Month MonthEnd of 0 1 1 2 3 6 7 12 18 Stage 1 Visit Description Post- Post-Antibody Post- Vaccin- Vacci- 6-Month Persis- Vaccin- Vaccination 1Tele- Vacci- ation 2 Vacci- nation 3 Follow- tence Tele- ation Follow-upphone nation Blood nation Blood up Blood phone 1 Visit Contact 2 Draw 3Draw Visit^(b) Draw^(b) Contact Follow-up Vaccination Phase PhaseVaccination & X  X X 30-minute observation^(c) Obtain 5-mL X^(d) X XX^(e) X blood sample Abbreviations: CRF = case report form; e-diary =electronic diary. ^(a)Visits 9 and 10 are not included in this primaryanalysis clinical study report. ^(b)Relative to Vaccination 3.^(c)Injection performed by unblinded administrator; acute reactionsassessed by blinded observer. Location of vaccination was noted in thesource, the CRF, and the e-diary. ^(d)Blood was collected beforevaccination and only after eligibility was confirmed. ^(e)Immunogenicityresults from blood draw at Visit 8 are not included in this primaryanalysis clinical study report. Source: Protocol schedule of activitiesfor Stage 2.

TABLE 24 Stage 2 Visit Schedule Visit Identifier 11 12 13 Time PeriodMonth 30 Month 42 Month 54 Visit Description ImmunogenicityImmunogenicity Immunogenicity 1 2 3 (Time After (24 Months) (36 Months)(48 Months) Vaccination 3) Obtain 5-mL X X X blood sample Source:Protocol schedule of activities for Stage 2.

Vaccines Administered: Bivalent rLP2086 (60 μg or 120 μg) wasadministered 3 times over the course of the study: the first vaccinationat Visit 1 (Month 0), second vaccination at Visit 4 (Month 2), and thirdvaccination at Visit 6 (Month 6). Bivalent rLP2086 was administered asan intramuscular injection into either the deltoid muscle oranterolateral thigh muscle. HAV vaccine was administered twice over thecourse of the study: the first vaccination at Visit 1 (Month 0) andthird vaccination at Visit 6 (Month 6). Saline was administered at thesecond vaccination (Month 2) time point. HAV vaccine/saline wasadministered as an intramuscular injection into either the deltoidmuscle or anterolateral thigh muscle. Only a third-party unblindedmedically qualified member of the study staff administered theinvestigational product. If muscle mass in the deltoid was not adequatefor intramuscular injection, then the thigh was the preferred injectionsite.

Immunogenicity Evaluations: To facilitate immunogenicity analysis,subjects had approximately 5 mL of blood collected at the following timepoints during Stage 1: before Vaccination 1 (Visit 1), 1 month afterVaccination 2 (Visit 5), 1 month after Vaccination 3 (Visit 7), 6 monthsafter Vaccination 3 (Visit 8), 12 months after Vaccination 3 (Visit 9).In total, 25 mL was collected over the 18-month period. Local/topicalanesthetic could be used prior to blood draws.

To determine duration of immune response, Stage 2 subjects will haveapproximately 5 mL of blood collected at the following time points: 2years after Vaccination 3, 3 years after Vaccination 3, and 4 yearsafter Vaccination 3. In total, 15 mL will be collected overapproximately 2.5 years.

For assessment of the immune response to bivalent rLP2086, functionalantibodies were analyzed in hSBAs with meningococcal serogroup Bstrains. The hSBA measures antibodies in human sera that result incomplement-dependent killing of the target meningococcal strain. Four(4) primary MnB test strains, PMB80 (A22), PMB2001 (A56), PMB2948 (B24),and PMB2707 (B44), each expressing a factor H binding protein (fHBP)variant heterologous to the vaccine component antigens, were used in thehSBAs for determination of the immunogenicity endpoints in this study.

Due to serum volume limitations, 2 of the primary strains (PMB80 [A22]and PMB2948 [B24]) were tested at each blood sampling time point forhalf of the subjects (in both age strata), and the other 2 primarystrains (PMB2001 [A56] and PMB2707 [B44]) were tested at each bloodsampling time point for the remaining half of the subjects.

Once all subjects completed enrollment (Visit 1), the independentstatistical center (ISC), a statistical team not involved in the conductof the study, provided 2 subject listings (randomly selected, 50% ofsubjects to be tested for PMB80 [A22]/PMB2948 [B24] and the remaining50% of subjects to be tested for PMB2001 [A56]/PMB2707 [B44]) to thesponsor's sample management team. Both listings followed the samerandomization ratio (2:1) and age-strata distribution as in the studydesign. The same strain pair (PMB80 [A22]/PMB2948 [B24] or PMB2001[A56]/PMB2707 [B44]) was tested across all visits for the same subjects.

Once testing for the primary analyses was completed, and if sufficientvolume of sera was available, additional testing to assess the immuneresponse to bivalent rLP2086 could be considered as follows: PMB80 (A22)and PMB2948 (B24) could be tested in serum samples from the 50% ofsubjects who received bivalent rLP2086 and whose serum samples wereoriginally tested for PMB2001 (A56) and PMB2707 (B44). Conversely,PMB2001 (A56) and PMB2707 (B44) could be tested in serum samples fromthe 50% of subjects who received bivalent rLP2086 and were originallytested for PMB80 (A22) and PMB2948 (B24). Testing for secondary strainscould be performed.

Statistical Methods:

The primary immunogenicity endpoints were:

-   -   Proportions of subjects achieving an hSBA titer ≥lower limit of        quantitation(LLOQ) 1 month after the third vaccination, for each        of the 4 primary MnB test strains in healthy toddlers 12 to <18        months of age at study entry.    -   Proportions of subjects achieving an hSBA titer ≥LLOQ 1 month        after the third vaccination, for each of the 4 primary MnB test        strains in healthy toddlers 18 to <24 months of age at study        entry.

All secondary immunogenicity endpoints for the entire study aredescribed here but this Example presents results for the immunogenicityendpoints applicable to Visits 1 to 7 only.

Subsequent endpoints will be analyzed.

The following endpoint applied to results in healthy subjects 12 monthsto <24 months of age (i.e., both age strata combined) at study entry:

-   -   Proportion of subjects with hSBA titers ≥LLOQ for each of the 4        primary MnB test strains 1 month after the third vaccination        with bivalent rLP2086 and 6, 12, 24, 36, and 48 months after the        third vaccination with bivalent rLP2016.

The following endpoints applied to results in healthy subjects 12 to <18months of age and 18 to <24 months of age at study entry, and in bothage strata combined:

-   -   Proportions of subjects with hSBA titers ≥LLOQ for each of the 4        primary MnB test strains at 1 month after the second vaccination        with bivalent rLP2086.    -   Proportions of subjects with hSBA titers ≥LLOQ, ≥1:4, ≥1:8,        ≥1:16, ≥1:32, ≥1:64, and ≥1:128 for each of the 4 primary MnB        strains at each applicable blood sampling visit.    -   hSBA geometric mean titers (GMTs) for each of the 4 primary MnB        test strains at each applicable blood sampling visit.

All of the exploratory endpoints specified below applied to hSBA resultsfrom all healthy subjects 12 to <18 months of age or 18 to <24 months ofage at study entry, and in both age strata combined, who receivedbivalent rLP2086 and were tested for the appropriate strain at theappropriate time point:

-   -   Proportions of subjects with hSBA titers ≥LLOQ, ≥1:4, ≥1:8,        ≥1:16, ≥1:32, ≥1:64, and ≥1:128 at each applicable blood        sampling time point.    -   hSBA GMTs for each of the 4 primary MnB strains at each        applicable blood sampling visit.    -   Proportions of subjects achieving at least a ≥4-fold increase in        hSBA titer from baseline to 1 month after the third vaccination        with bivalent rLP2086 for each of the 4 primary test strains:        -   For subjects with a baseline hSBA titer below the limit of            detection (LOD) or an hSBA titer of <1:4, a ≥4-fold response            was defined as an hSBA titer of ≥1:16.        -   For subjects with a baseline hSBA titer of ≥LOD (ie, hSBA            titer of ≥1:4) and <LLOQ, a ≥4-fold response was defined as            an hSBA titer of 4 times the LLOQ.        -   For subjects with a baseline hSBA titer of ≥LLOQ, a ≥4-fold            response was defined as an hSBA titer of ≥4 times the            baseline titer.

The following endpoints were planned if there had been sufficient seraavailable to test each subject for all 4 primary strains and/or to testsubjects for the secondary strains.

-   -   Proportions of subjects achieving an hSBA titer ≥LLOQ for all 4        primary test strains (PMB80 [A22], PMB2948 [B24], PMB2001 [A56],        and PMB2707 [B44]) combined, 1 month after the third vaccination        with bivalent rLP2086. This applied only to those subjects who        had all 4 primary strains tested.    -   Additional exploratory assays to test hSBA on the secondary MnB        strains as follows:        -   hSBA GMTs to each secondary MnB strain tested, at 1 month            after the second and third vaccinations and/or at each blood            sampling time point thereafter.        -   Proportions of subjects with an hSBA titer ≥LLOQ, to each            secondary MnB strain at 1 month after the second and third            vaccinations and/or at each blood sampling time point            thereafter.

Analysis of Primary Endpoints

The primary analysis for the primary objectives was the proportion ofsubjects with an hSBA titer ≥LLOQ 1 month after the third vaccination,for each of the 4 primary MnB test strains in healthy toddlers aged 12to <18 months, and 18 to <24 months, at study entry respectively. Theevaluable immunogenicity population was used for this summary and bothpercentages and confidence intervals (CIs) are displayed.

Analysis of Secondary Endpoints

All of the analyses performed on the mITT population were considered assecondary analyses.

Secondary analyses also included percent of subjects with hSBA titers≥LLOQ for each of the 4 primary MnB test strains 1 month following thethird vaccination for both the evaluable immunogenicity population andfor the mITT population.

For this Example, the percentage of subjects with hSBA titers ≥LLOQ foreach of the 4 primary MnB test strains 1 month following the secondvaccination and 1 month after the third vaccination were analyzed usingboth the evaluable immunogenicity population and the mITT population.For the final analysis, the percentage of subjects with hSBA titers≥LLOQ for each of the 4 primary MnB test strains 6, 12, 24, 36, and 48months after the third vaccination will be analyzed using both theevaluable immunogenicity population and the mITT population.

The percentage of subjects with hSBA titers ≥LLOQ, 1:4, 1:8, 1:16, 1:32,1:64, 1:128 for each of the 4 primary MnB test strains at eachapplicable blood sampling visit were analyzed using both the evaluableimmunogenicity population and the mITT population. The GMTs for each ofthe primary MnB test strains at each applicable blood sampling visitwere summarized for the evaluable immunogenicity population and the mITTpopulation.

Analysis of Exploratory Endpoints

The proportion of subjects achieving at least a ≥4-fold increase in hSBAtiter from baseline to 1 month after the second and third vaccinationswith bivalent rLP2086 was summarized for the evaluable immunogenicitypopulation and the mITT population.

Subgroup Analysis

Some immunogenicity and safety endpoints were descriptively summarizedby sex, by country, and within age strata.

Results

Subject Disposition and Demography:

A total of 396 subjects 12 to <24 months of age were randomized in thisstudy. Of the subjects randomized, a total of 44 subjects received 60 μgof bivalent rLP2086, 220 subjects received 120 μg of bivalent rLP2086,and 132 subjects received HAV vaccine/saline. There were 198 subjectsrandomized in each of the age strata (12 to <18 months and 18 to <24months).

Of the 396 randomized subjects, 385 (97.2%) subjects completed thevaccination phase (from the first study vaccination [Visit 1] through 1month after Vaccination 3 [Visit 7]) of the study. A total of 386(97.5%) subjects completed the follow-up phase (from Visit 7 to 6 monthsafter Vaccination 3 [Visit 8]). Overall, a total of 381 (96.2%) subjectscompleted all study visits up to the 6-month follow-up visit (Visit 8)and completion status was similar for each age stratum (189 [95.5%]subjects and 192 [97.0%] subjects in the 12 to <18 months and 18 to <24months age strata, respectively).

Overall, 52.8% of subjects were female, and the majority of the subjectswere white (94.4%) and non-Hispanic/non-Latino (99.5%). The mean age(SD) at first vaccination was 17.3 (3.61) months (range of 12 to 23months). Demographic characteristics were generally similar among thevaccine groups.

The characteristics of ITT and mITT populations were similar to thecharacteristics of the safety population.

Immunogenicity Results:

The primary objectives of this study were to describe the immuneresponse to bivalent rLP2086 as measured by hSBA against 4 primary MnBtest strains, 2 expressing an LP2086 subfamily A protein and 2expressing an LP2086 subfamily B protein, measured 1 month after thethird vaccination in healthy subjects 12 to <18 months of age and 18 to<24 months of age. The description of immune responses for the combinedage stratum (12 to <24 months) was a secondary objective. The primaryendpoints for the primary objectives were the proportions of subjects ineach age stratum achieving hSBA titers ≥LLOQ for each of the 4 primaryMnB strains 1 month after the third vaccination.

A robust immune response was observed at both dose levels for toddlers12 to <18 months of age and for toddlers 18 to <24 months of age, aswell as for the combined age stratum (12 to <24 months) 1 month afterthe third dose of bivalent rLP2086, as confirmed by the proportion ofsubjects achieving an hSBA titer ≥LLOQ (1:8 for A56, B24 and B44; 1:16for A22) for each of the 4 primary MnB test strains. For the 60-μggroup, the proportion of subjects achieving an hSBA titer ≥LLOQ rangedfrom 88.9% to 100.0% for the younger toddlers (12 to <18 months) andfrom 81.8% to 100.0% for the older toddlers (18 to <24 months) after 3doses. For the 120-μg group, the proportion of subjects achieving anhSBA titer ≥LLOQ ranged from 71.1% to 100.0% for toddlers 12 to <18months of age and from 72.0% to 100.0% for toddlers 18 to <24 months ofage after 3 doses. For the combined age stratum the proportion ofsubjects achieving an hSBA titer ≥LLOQ for each of the 4 primary MnBtest strains 1 month after the third vaccination ranged from 85.0% to100.0% for the 60-μg group and from 71.6% to 100.0% for the 120-μggroup. These findings are further supported by increases in GMTs (rangefrom 4.0 to 8.5 at baseline to 15.1 to 171.4 at 1 month afterVaccination 3) and in the proportion of subjects achieving an hSBA titer≥1:4 (71.1% to 100.0%) or ≥1:16 (63.6% to 100.0%) against each of the 4primary MnB test strains after 3 doses of bivalent rLP2086 compared tobaseline across both age strata and dose levels. Additionally, theproportion of subjects for the combined age stratum achieving an hSBAfold rise 4 from baseline to 1 month after the third vaccination foreach of the 4 primary MnB test strains ranged from 67.4% to 100.0% forboth dose levels. In conclusion, 3 doses of either 60 μg or 120 μg ofbivalent rLP2086 administered on a 0-, 2-, 6-month schedule, inducedrobust immune responses in toddlers 12 to <24 months of age (bothindividual and combined age strata).

The secondary objective of the study was to describe immune responses 1month after the second dose of bivalent rLP2086, as assessed by ≥LLOQresponses, defined hSBA titers, and hSBA GMTs for the 2 age strata andthe combined age stratum. For the combined age stratum, the proportionof subjects achieving an hSBA titer ≥LLOQ after the second dose ofbivalent rLP2086 (administered 2 months after the first dose) rangedfrom 57.9% to 94.7% for subjects in the 60-μg group and from 33.7% to100.0% for subjects in the 120-μg group. Similar results were obtainedfor the 2 individual age strata with no clinically meaningfuldifferences between the younger and older age strata. These findings aresupported by increases in GMTs (range 7.2 to 110.6) over baseline and inthe proportion of subjects achieving an hSBA titer ≥1:4 (36.0% to100.0%) or ≥1:16 (32.6% to 100%) against each of the 4 primary MnB teststrains after 2 doses of bivalent rLP2086 compared to baseline acrossboth dose levels for the combined age stratum. Similar results wereobtained for the 2 individual age strata. Additionally, the proportionof subjects for the combined age stratum achieving an hSBA fold rise 4from baseline to 1 month after the second vaccination for each of the 4primary MnB test strains ranged from 30.2% to 98.9%. In conclusion, 2doses of either 60 μg or 120 μg of bivalent rLP2086 administered 2months apart induced immune responses in toddlers 12 to <24 months ofage (both individual and combined age strata). In summary, at both the60-μg and 120-μg dose levels, bivalent rLP2086 given as 3 doses on a 0-,2-, and 6-month schedule elicits a robust immune response among toddlers12 to <24 months of age with protective antibody titers achieved asmeasured by hSBA in a high proportion of subjects after the third dose.

Conclusions:

In conclusion, the 60-μg and 120-μg dose levels of bivalent rLP2086 whenadministered to toddlers 12 to <24 months of age on a 0-, 2-, and6-month schedule elicit protective antibody titers after the third doseas measured by hSBAs. The vaccine, as administered in this study, wassafe and well tolerated with an acceptable safety profile for toddlers12 to <24 months of age.

EXAMPLE 21: A Phase 2, Randomized, Controlled, Observer-Blinded StudyConducted to Describe the Immunogenicity, Safety, and Tolerability of aNeisseria meningitidis Serogroup B Bivalent Recombinant Lipoprotein 2086Vaccine (Bivalent rLP2086) When Administered to Healthy Toddlers Aged 12to <18 Months or 18 to <24 Months (B1971035-CSR)

This Phase 2 study was conducted in 2 stages. Stage 1 was designed andconducted to assess the safety, tolerability, and immunogenicity ofbivalent rLP2086 in healthy toddlers aged 12 to <24 months through 12months after the last study vaccination vaccination (Visit 10). Stage 1was composed of a sentinel-enrollment phase with 4 sentinel cohorts andan expanded-enrollment phase. In the sentinel cohorts, bivalent rLP2086was administered at 2 dose levels (60 μg and 120 μg) in 2 age strata (12to <15 months and 18 to <24 months). Selection of dose level for theexpanded-enrollment phase was based on an internal review committee(IRC) review of the safety profile of the 2 dose levels. Stage 2 wasplanned to assess the duration of the immune response up through 4 yearsafter the final study vaccination.

Primary Immunogenicity Objectives

-   -   To describe the immune response as measured by hSBA performed        with 4 primary MnB strains, 2 expressing an LP2086 subfamily A        protein and 2 expressing an LP2086 subfamily B protein, measured        1 month after the third vaccination with bivalent rLP2086, in        healthy toddlers aged 12 to <18 months at study entry.    -   To describe the immune response as measured by hSBA performed        with 4 primary MnB strains, 2 expressing an LP2086 subfamily A        protein and 2 expressing an LP2086 subfamily B protein, measured        1 month after the third vaccination with bivalent rLP2086, in        healthy toddlers aged 18 to <24 months at study entry.

Secondary Immunogenicity Objectives

-   -   To describe the immune response as measured by hSBA performed        with 4 primary MnB test strains, 2 expressing an LP2086        subfamily A protein and 2 expressing an LP2086 subfamily B        protein, measured 1 month after the third vaccination with        bivalent rLP2086, in healthy toddlers aged 12 to <24 months at        study entry (ie, both age strata combined).    -   To describe the immune response as measured by hSBA performed        with 4 primary MnB test strains, 2 expressing an LP2086        subfamily A protein and 2 expressing an LP2086 subfamily B        protein, measured 1 month after the second vaccination and 6,        12, 24, 36, and 48 months after the third vaccination in healthy        toddlers aged 12 to <18 months and 18 to <24 months at study        entry, and in both age strata combined.

Exploratory Immunogenicity Objectives

-   -   To further describe the immune response as measured by hSBA        performed with 4 primary MnB test strains, 2 expressing an        LP2086 subfamily A protein and 2 expressing an LP2086 subfamily        B protein, measured 1 month after the second vaccination and 1,        6, 12, 24, 36, and 48 months after the third vaccination with        bivalent rLP2086 in healthy toddlers aged 12 to <18 months and        18 to <24 months at study entry, and in both age strata        combined.    -   To further describe the immune response as measured by hSBA to        secondary MnB test strains expressing LP2086 subfamily A and B        proteins, at 1 month after the second vaccination and 1, 6, 12,        24, 36, and 48 months after the third vaccination in healthy        toddlers aged 12 to <18 months and 18 to <24 months at study        entry, and in both age strata combined.

Investigational Plan

Overall Study Design and Plan

The study is a Phase 2, randomized, active-controlled, observer-blinded,sponsor-unblinded, multicenter study in which approximately 396 healthytoddlers stratified by age, 12 to <18 months or 18 to <24 months old,were randomly assigned in a 2:1 ratio to receive bivalent rLP2086(either of 2 dose levels [60 μg or 120 μg]) or a licensed pediatric HAVvaccine (0.5 mL)/sterile saline solution for injection (0.5-mL of 0.85%sodium chloride). The study was conducted in 2 stages. Stage 1 assessedvaccine immunogenicity, safety, and tolerability across 2 phases: asentinel-enrollment phase and an expanded-enrollment phase. Stage 2assessed the duration of the immune response to bivalent rLP2086. TheStage 1 sentinel-enrollment phase was planned to include a total of 4sentinel cohorts: 2 age strata for each dose level (60 μg or 120 μg) ofbivalent rLP2086. The younger-aged sentinel cohorts were composed ofsubjects aged 12 to <15 months and the older-aged sentinel cohorts werecomposed of subjects aged 18 to <24 months. Each of the 4 sentinelcohorts was planned to enroll approximately 33 subjects. Thesentinel-enrollment phase was staggered with reviews by an IRC atpre-specified points and stopping rules applied. The 120-μg dose levelsentinel cohorts did not proceed until the 60-μg dose level wasevaluated by the IRC as safe and tolerable in the sentinel cohort of thesame age. The younger-aged 120-μg dose level sentinel cohort did notproceed until this dose level was evaluated by the IRC as safe andtolerable in the older-aged 120-μg dose level sentinel cohort.

Prior to the Stage 1 expanded-enrollment phase, the IRC reviewed allpost-Vaccination 1, 7-day e-diary and SAE data obtained from sentinelsubjects. Based on the review, the IRC selected the 120-μg bivalentrLP2086 dose level to be studied in the Stage 1 expanded-enrollmentphase for both age strata. The younger-aged expanded-enrollment cohort(enrolling an additional 132 subjects) was extended to subjects aged 12to <18 months and stratified by age into 2 subsets: aged 12 to <15months and aged 15 to <18 months. The older-aged expanded enrollmentcohort (enrolling an additional 132 subjects) enrolled subjects aged 18to <24 months during the expanded-enrollment phase. The total studyduration for subjects completing only Stage 1 will be approximately 18months.

Stage 2 includes only those subjects randomly assigned to bivalentrLP2086 (irrespective of dose level). The total study duration forsubjects who complete Stage 2 will be approximately 4.5 years (54months).

Bivalent rLP2086 was administered at Months 0, 2, and 6 (Visits 1, 4,and 6). Pediatric HAV vaccine was administered at Months 0 and 6 (Visits1 and 6), and saline was administered at Month 2 (Visit 4) to maintainthe study blind.

Discussion of Study Design, Including Choice of Control Groups

This 2-stage study evaluated the safety, tolerability, andimmunogenicity of bivalent rLP2086 at 2 dose levels (60 μg and 120 μg)in healthy toddlers aged 12 to <24 months. HAV vaccine (at Months 0 and6) was chosen as the control in this study. In comparison to otherrecommended vaccines for this age group, HAV vaccine has a bettertolerability profile. In addition, HAV vaccine confers a benefit tosubjects who might be at increased risk for hepatitis A viral infectioneither during future travel or during other exposures. The generallyrecommended regimen for HAV vaccine is 2 doses at Months 0 and 6. Inthis study, saline was given at Month 2 to maintain the study blind.

Vaccines Administered

Bivalent rLP2086 (60 μg or 120 μg) was administered 3 times over thecourse of the study: the first vaccination at Visit 1 (Month 0), secondvaccination at Visit 4 (Month 2), and third vaccination at Visit 6(Month 6). Bivalent rLP2086 was administered as an intramuscularinjection into either the deltoid muscle or anterolateral thigh muscle.HAV vaccine was administered twice over the course of the study: thefirst vaccination at Visit 1 (Month 0) and third vaccination at Visit 6(Month 6). Saline was administered at the second vaccination (Month 2)time point. HAV vaccine/saline was administered as an intramuscularinjection into either the deltoid muscle or anterolateral thigh muscle.Only a third-party unblinded medically qualified member of the studystaff administered the investigational product. If muscle mass in thedeltoid was not adequate for intramuscular injection, then the thigh wasthe preferred injection site. Site of administration (eg, left/rightarm/thigh) was noted in the source notes and on the CRF.

Identity of Investigational Product(s)

Bivalent rLP2086 (containing either 30 μg [60-μg dose level] or 60 μg[120-μg dose level] each of a purified subfamily A and subfamily BrLP2086 protein, adsorbed to aluminum in a sterile buffered isotonicsuspension) was provided in a 0.5-mL dose for injection.

A licensed pediatric HAV vaccine was provided in a 0.5-mL dose forinjection.

The placebo was sterile saline for injection (0.85% sodium chloride)supplied as a 0.5-mL dose.

The investigational products (bivalent rLP2086, HAV vaccine, and saline)were provided by the sponsor to each study site. Study vaccines werepacked and labeled as investigational product in accordance with currentguidelines and applicable local and legal regulatory requirements. Eachinvestigational product was labeled with a unique kit number.

Selection of Vaccination Regimen

Bivalent rLP2086 (either 60 μg or 120 μg) was administered on a Month 0,2, and 6 schedule. The control group of subjects received HAV vaccine atMonth 0 and Month 6 and an injection of saline at Month 2 to maintainthe study blind.

Bivalent rLP2086 Serum Bactericidal Assay—Primary Test Strains

For assessment of the immune response to bivalent rLP2086, functionalantibodies were analyzed in hSBAs with meningococcal serogroup Bstrains. The hSBA measures antibodies in human sera that mediatecomplement-dependent killing of the target meningococcal strain. Four(4) primary MnB test strains, PMB80 (A22), PMB2001 (A56), PMB2948 (B24),and PMB2707 (B44), each expressing a factor H binding protein (fHBP)variant heterologous to the vaccine component antigens, were used in thehSBAs for determination of the immunogenicity endpoints in this study.

Due to serum volume limitations, 2 of the primary strains (PMB80 [A22]and PMB2948 [B24]) were tested at each blood sampling time point forhalf of the subjects (in both age strata), and the other 2 primarystrains (PMB2001 [A56] and PMB2707 [B44]) were tested at each bloodsampling time point for the remaining half of the subjects.

Once all subjects completed enrollment (Visit 1), the independentstatistical center (ISC), a statistical team not involved in the conductof the study, provided 2 subject listings (randomly selected, 50% ofsubjects to be tested for PMB80 [A22]/PMB2948 [B24] and the remaining50% of subjects to be tested for PMB2001 [A56]/PMB2707 [B44]) to thesponsor's sample management team. Both listings followed the samerandomization ratio (2:1) and age-strata distribution as in the studydesign. The same strain pair (PMB80 [A22]/PMB2948 [B24] or PMB2001[A56]/PMB2707 [B44]) was tested across all visits for the same subjects.

Additional Assays

Once testing for the primary analyses was completed, and if sufficientvolume of sera was available, additional testing to assess the immuneresponse to bivalent rLP2086 could be considered as follows: PMB80 (A22)and PMB2948 (B24) could be tested in serum samples from the 50% ofsubjects who received bivalent rLP2086 and whose serum samples wereoriginally tested for PMB2001 (A56) and PMB2707 (B44). Conversely,PMB2001 (A56) and PMB2707 (B44) could be tested in serum samples fromthe 50% of subjects who received bivalent rLP2086 and were originallytested for PMB80 (A22) and PMB2948 (B24). Testing for secondary strainscould be performed.

Immunogenicity Analysis

Comparisons of Interest and Endpoints—Primary Immunogenicity Endpoints

The primary immunogenicity endpoints were:

-   -   Proportions of subjects achieving an hSBA titer ≥lower limit of        quantitation(LLOQ) 1 month after the third vaccination, for each        of the 4 primary MnB test strains in healthy toddlers 12 to <18        months of age at study entry.    -   Proportions of subjects achieving an hSBA titer ≥LLOQ 1 month        after the third vaccination, for each of the 4 primary MnB test        strains in healthy toddlers 18 to <24 months of age at study        entry.

Comparisons of Interest and Endpoints—Secondary Immunogenicity Endpoints

All secondary immunogenicity endpoints for the entire study aredescribed here but this Example will present results for theimmunogenicity endpoints applicable to Visits 1 to 7 (Vaccination 1 to 3months after Vaccination 3) only.

The following endpoint applied to results in healthy subjects 12 monthsto <24 months of age (ie, both age strata combined) at study entry:

-   -   Proportion of subjects with hSBA titers ≥LLOQ for each of the 4        primary MnB test strains 1 month after the third vaccination        with bivalent rLP2086 and 6, 12, 24, 36, and 48 months after the        third vaccination with bivalent rLP2016.

The following endpoints applied to results in healthy subjects 12 to <18months of age and 18 to <24 months of age at study entry, and in bothage strata combined:

-   -   Proportions of subjects with hSBA titers ≥LLOQ for each of the 4        primary MnB test strains at 1 month after the second vaccination        with bivalent rLP2086.    -   Proportions of subjects with hSBA titers ≥LLOQ, ≥1:4, ≥1:8,        ≥1:16, ≥1:32, ≥1:64, and ≥1:128 for each of the 4 primary MnB        strains at each applicable blood sampling visit.    -   hSBA geometric mean titers (GMTs) for each of the 4 primary MnB        test strains at each applicable blood sampling visit.

Exploratory Immunogenicity Endpoints

All of the exploratory endpoints specified below applied to hSBA resultsfrom all healthy subjects 12 to <18 months of age or 18 to <24 months ofage at study entry, and in both age strata combined, who receivedbivalent rLP2086 and were tested for the appropriate strain at theappropriate time point:

-   -   Proportions of subjects with hSBA titers ≥LLOQ, ≥1:4, 1:8, 1:16,        ≥1:32, ≥1:64, and ≥1:128 at each applicable blood sampling time        point.    -   hSBA GMTs for each of the 4 primary MnB strains at each        applicable blood sampling visit.    -   Proportions of subjects achieving at least a 4-fold increase in        hSBA titer from baseline to 1 month after the third vaccination        with bivalent rLP2086 for each of the 4 primary test strains:        -   For subjects with a baseline hSBA titer below the limit of            detection (LOD) or an hSBA titer of <1:4, a ≥4-fold response            was defined as an hSBA titer of ≥1:16.        -   For subjects with a baseline hSBA titer of ≥LOD (ie, hSBA            titer of ≥1:4) and <LLOQ, a ≥4-fold response was defined as            an hSBA titer of ≥4 times the LLOQ.        -   For subjects with a baseline hSBA titer of ≥LLOQ, a ≥4-fold            response was defined as an hSBA titer of ≥4 times the            baseline titer.

The following endpoints were considered if there had been sufficientsera available to test each subject for all 4 primary strains and/or totest subjects for the secondary strains.

-   -   Proportions of subjects achieving an hSBA titer ≥LLOQ for all 4        primary test strains (PMB80 [A22], PMB2948 [B24], PMB2001 [A56],        and PMB2707 [B44]) combined, 1 month after the third vaccination        with bivalent rLP2086. This applied only to those subjects who        had all 4 primary strains tested.    -   Additional exploratory assays to test hSBA on the secondary MnB        strains as follows:        -   hSBA GMTs to each secondary MnB strain tested, at 1 month            after the second and third vaccinations and/or at each blood            sampling time point thereafter.        -   Proportions of subjects with an hSBA titer ≥LLOQ, to each            secondary MnB strain at 1 month after the second and third            vaccinations and/or at each blood sampling time point            thereafter.

Analysis Populations

Modified Intent-to-Treat Population

All randomized subjects who had at least 1 valid and determinate assayresult related to a proposed analysis were included in the modifiedintent-to-treat (mITT) population. This analysis set was for theimmunogenicity analysis. Subjects were analyzed according to theinvestigational product to which they were randomized in the analysis ofthe mITT population.

Methods of Analysis

The control group and each dose-level group from different cohortswithin the same age stratum were pooled for analysis. All of theimmunogenicity analyses were summarized for each age stratum separately,as well as for the overall population.

This was not a hypothesis-testing study; thus, an estimation approachwas used to assess the primary, secondary, and exploratory objectives inthis study.

The LLOQ was 1:16 for PMB80 (A22), 1:8 for PMB2001 (A56), 1:8 forPMB2707 (B44), and 1:8 for PMB2948 (B24).

For the calculation of GMTs, hSBA results below the LLOQ were set to0.5×LLOQ for the primary analysis.

Analysis of Primary Endpoints

The primary analysis for the primary objectives was the proportion ofsubjects with an hSBA titer ≥LLOQ 1 month after the third vaccination,for each of the 4 primary MnB test strains in healthy toddlers aged 12to <18 months, and 18 to <24 months, at study entry respectively.

The evaluable immunogenicity population was used for this summary andboth percentages and confidence intervals (CIs) are displayed.

Analysis of Secondary Endpoints

All of the analyses performed on the mITT population were considered assecondary analyses.

Analyses for secondary endpoints also included percent of subjects withhSBA titers ≥LLOQ for each of the 4 primary MnB test strains 1 monthfollowing the third vaccination for both the evaluable immunogenicitypopulation and for the mITT population.

For this Example, the percentage of subjects with hSBA titers ≥LLOQ foreach of the 4 primary MnB test strains at 1 month after Vaccination 2and 1 month after Vaccination 3 were analyzed using both the evaluableimmunogenicity population and the mITT population and presented in thisreport. The same analysis is planned for subsequent time points (6, 12,24, 36, and 48 months after the third vaccination).

The percentage of subjects with hSBA titers ≥LLOQ, 1:4, 1:8, 1:16, 1:32,1:64, 1:128 for each of the 4 primary MnB test strains at 1 month afterVaccination 2 and 1 month after Vaccination 3 were analyzed using boththe evaluable immunogenicity population and the mITT population andpresented in this report. The same analysis is planned for subsequenttime points (6, 12, 24, 36, and 48 months after Vaccination 3).

The GMTs for each of the primary MnB test strains at 1 month followingthe second vaccination and 1 month after the third vaccination weresummarized for the evaluable immunogenicity population and the mITTpopulation and presented in this report. The same analysis is plannedfor subsequent time points (6, 12, 24, 36, and 48 months afterVaccination 3).

Analysis of Exploratory Endpoints

The proportion of subjects achieving at least a 4-fold increase in hSBAtiter from baseline to 1 month after the second and third vaccinationswith bivalent rLP2086 was summarized for the evaluable immunogenicitypopulation and the mITT population.

Reverse Cumulative Distribution Curves

The empirical reverse cumulative distribution curves (RCDCs) were alsoassessed for each of the 4 primary MnB test strains and at 1 month afterVaccination 2 and 1 month after Vaccination 3 for the evaluableimmunogenicity population.

Immunogenicity Evaluation

Populations Analyzed

The evaluable immunogenicity population was the primary analysispopulation for the immunogenicity analyses. The mITT population was usedas a supportive immunogenicity population for the immunogenicityanalyses.

A total of 348 (87.9%) subjects were included in the evaluableimmunogenicity population, and 48 (12.1%) subjects were excluded fromthe evaluable immunogenicity population. Subjects could have beenexcluded from the immunogenicity populations for more than 1 reason. Atotal of 31 (7.8%) subjects were excluded from the evaluableimmunogenicity population because they did not have scheduledprevaccination or postvaccination blood drawn (includes subjects who didnot have samples taken and subjects with samples taken outside of theprotocol-specified window), 13 (3.3%) subjects were not eligible orbecame ineligible for the study before or at the 1-monthpost-Vaccination 3 visit, 11 (2.8%) subjects did not receive vaccine asrandomized at all vaccination visits, and 16 (4.0%) subjects receivedprohibited vaccines or treatments. A total of 84.3% of subjects 12 to<18 months of age and 91.4% of subjects 18 to <24 months of age wereincluded in the evaluable immunogenicity population.

Immunogenicity Results

The primary analysis results including all of the immunogenicity datathrough 1 month after Vaccination 3 (Visit 7) are provided in thefollowing sections. The analysis of immunogenicity endpoints was basedon hSBA results for strains PMB2001 (A56) and PMB2707 (B44) for half ofthe subjects, and strains PMB80 (A22) and PMB2948 (B24) for theremaining half.

Primary and Secondary Endpoints

Proportion of Subjects Achieving an hSBA Titer ≥LLOQ

The primary immunogenicity endpoints were the proportions of subjects 12to <18 months of age (at study entry), and 18 to <24 months of age (atstudy entry), achieving an hSBA titer ≥LLOQ for each of the 4 primaryMnB test strains 1 month after the third vaccination with bivalentrLP2086. The proportion of all subjects in the combined age stratumachieving an hSBA titer ≥LLOQ for each of the 4 primary MnB test strains1 month after the third vaccination with bivalent rLP2086, along withthe proportion of subjects in the individual and combined age strataachieving an hSBA titer ≥LLOQ for each of the 4 primary MnB test strains1 month after the second vaccination with bivalent rLP2086, weresecondary endpoints.

The proportion of subjects in each age stratum achieving an hSBA titer≥LLOQ for each of the 4 primary MnB test strains is presented in Tablefor the evaluable immunogenicity population.

For the combined age stratum the proportion of subjects achieving anhSBA titer ≥LLOQ at baseline was 0.0% and 3.1% for PMB80 (A22); 0.0% and1.1% for PMB2001 (A56); 4.8% and 2.1% for PMB2948 (B24); and 0.0% and1.1% for PMB2707 (B44) for the 60-μg and 120-μg groups, respectively.

At 1 month after Vaccination 2, the proportion of subjects achieving anhSBA titer ≥LLOQ for the 12 to <18 months age stratum was 90.0% and64.4% for PMB80 (A22); 100.0% and 100.0% for PMB2001 (A56); 70.0% and23.8% for PMB2948 (B24); and 77.8% and 72.3% for PMB2707 (B44) for the60-μg and 120-μg groups, respectively. For the 18 to <24 months agestratum, the proportion of subjects achieving an hSBA titer ≥LLOQ was66.7% and 84.0% for PMB80 (A22); 90.0% and 100.0% for PMB2001 (A56);44.4% and 43.2% for PMB2948 (B24); and 60.0% and 63.8% for PMB2707 (B44)for the 60-μg and 120-μg groups, respectively. Overall, for the combinedage stratum the proportion of subjects achieving an hSBA titer ≥LLOQ at1 month after Vaccination 2 was 78.9% and 74.7% for PMB80 (A22); 94.7%and 100.0% for PMB2001 (A56); 57.9% and 33.7% for PMB2948 (B24); and68.4% and 68.1% for PMB2707 (B44) for the 60-μg and 120-μg groups,respectively.

At 1 month after Vaccination 3, the proportion of subjects achieving anhSBA titer ≥LLOQ for the 12 to <18 months age stratum was 88.9% and91.1% for PMB80 (A22); 100.0% and 100.0% for PMB2001 (A56); 88.9% and71.1% for PMB2948 (B24); and 88.9% and 87.2% for PMB2707 (B44) for the60-μg and 120-μg groups, respectively. In the 18 to <24 months agestratum, the proportion of subjects achieving an hSBA titer ≥LLOQ was90.9% and 88.2% for PMB80 (A22); 100.0% and 100.0% for PMB2001 (A56);81.8% and 72.0% for PMB2948 (B24); and 90.0% and 85.1% for PMB2707 (B44)for the 60-μg and 120-μg groups, respectively. Overall, for the combinedage stratum the proportion of subjects achieving an hSBA titer ≥LLOQ at1 month after Vaccination 3 was 90.0% and 89.6% for PMB80 (A22); 100.0%and 100.0% for PMB2001 (A56); 85.0% and 71.6% for PMB2948 (B24); and89.5% and 86.2% for PMB2707 (B44) for the 60-μg and 120-μg groups,respectively.

In general, the proportion of subjects in the HAV/saline group achievingan hSBA titer ≥LLOQ did not change over time compared to baseline (Table25).

Results for the mITT population were similar to those of the evaluableimmunogenicity population.

Subgroup analyses of the proportion of subjects achieving an hSBA titer≥LLOQ for each of the 4 primary MnB test strains were assessed for theevaluable immunogenicity population by sex and country. There were noclinically important differences observed in the subgroup analysesperformed.

TABLE 25 Subjects With hSBA Titer ≥LLOQ for Primary Strains—EvaluableImmunogenicity Population Strain (Variant) Vaccine Group (as Randomized)Sampling Time Point 60 μg rLP2086 120 μg rLP2086 HAV/Saline Age StrataN^(a) n^(b) (%) (95% CI)^(c) N^(a) n^(b) (%) (95% CI)^(c) N^(a) n^(b)(%) (95% CI)^(c) PMB80 (A22) Before Vaccination 1 12 to <24 Months 20 0(0.0) (0.0, 16.8) 97 3 (3.1) (0.6, 8.8) 61 1 (1.6) (0.0, 8.8) 12 to <18Months 9 0 (0.0) (0.0, 33.6) 46 1 (2.2)  (0.1, 11.5) 31 0 (0.0)  (0.0,11.2) 18 to <24 Months 11 0 (0.0) (0.0, 28.5) 51 2 (3.9)  (0.5, 13.5) 301 (3.3)  (0.1, 17.2) 1 Month after Vaccination 2 12 to <24 Months 19 15(78.9) (54.4, 93.9) 95 71 (74.7) (64.8, 83.1) 59 1 (1.7) (0.0, 9.1) 12to <18 Months 10 9 (90.0) (55.5, 99.7) 45 29 (64.4) (48.8, 78.1) 30 0(0.0)  (0.0, 11.6) 18 to <24 Months 9 6 (66.7) (29.9, 92.5) 50 42 (84.0)(70.9, 92.8) 29 1 (3.4)  (0.1, 17.8) 1 Month after Vaccination 3 12 to<24 Months 20 18 (90.0) (68.3, 98.8) 96 86 (89.6) (81.7, 94.9) 60 3(5.0)  (1.0, 13.9) 12 to <18 Months 9 8 (88.9) (51.8, 99.7) 45 41 (91.1)(78.8, 97.5) 31 1 (3.2)  (0.1, 16.7) 18 to <24 Months 11 10 (90.9)(58.7, 99.8) 51 45 (88.2) (76.1, 95.6) 29 2 (6.9)  (0.8, 22.8) PMB2001(A56) Before Vaccination 1 12 to <24 Months 19 0 (0.0) (0.0, 17.6) 95 1(1.1) (0.0, 5.7) 53 0 (0.0) (0.0, 6.7) 12 to <18 Months 9 0 (0.0) (0.0,33.6) 46 0 (0.0) (0.0, 7.7) 24 0 (0.0)  (0.0, 14.2) 18 to <24 Months 100 (0.0) (0.0, 30.8) 49 1 (2.0)  (0.1, 10.9) 29 0 (0.0)  (0.0, 11.9) 1Month after Vaccination 2 12 to <24 Months 19 18 (94.7) (74.0, 99.9) 9595 (100.0)  (96.2, 100.0) 52 0 (0.0) (0.0, 6.8) 12 to <18 Months 9 9(100.0)  (66.4, 100.0) 47 47 (100.0)  (92.5, 100.0) 23 0 (0.0)  (0.0,14.8) 18 to <24 Months 10 9 (90.0) (55.5, 99.7) 48 48 (100.0)  (92.6,100.0) 29 0 (0.0)  (0.0, 11.9) 1 Month after Vaccination 3 12 to <24Months 19 19 (100.0)  (82.4, 100.0) 95 95 (100.0)  (96.2, 100.0) 54 1(1.9) (0.0, 9.9) 12 to <18 Months 9 9 (100.0)  (66.4, 100.0) 47 47(100.0)  (92.5, 100.0) 24 0 (0.0)  (0.0, 14.2) 18 to <24 Months 10 10(100.0)  (69.2, 100.0) 48 48 (100.0)  (92.6, 100.0) 30 1 (3.3)  (0.1,17.2) PMB2948 (B24) Before Vaccination 1 12 to <24 Months 21 1 (4.8) (0.1, 23.8) 97 2 (2.1) (0.3, 7.3) 61 1 (1.6) (0.0, 8.8) 12 to <18Months 10 0 (0.0)  (0.0, 30.8) 46 1 (2.2)  (0.1, 11.5) 31 0 (0.0)  (0.0,11.2) 18 to <24 Months 11 1 (9.1)  (0.2, 41.3) 51 1 (2.0)  (0.0, 10.4)30 1 (3.3)  (0.1, 17.2) 1 Month after Vaccination 2 12 to <24 Months 1911 (57.9) (33.5, 79.7) 86 29 (33.7) (23.9, 44.7) 59 1 (1.7) (0.0, 9.1)12 to <18 Months 10 7 (70.0) (34.8, 93.3) 42 10 (23.8) (12.1, 39.5) 30 0(0.0)  (0.0, 11.6) 18 to <24 Months 9 4 (44.4) (13.7, 78.8) 44 19 (43.2)(28.3, 59.0) 29 1 (3.4)  (0.1, 17.8) 1 Month after Vaccination 3 12 to<24 Months 20 17 (85.0) (62.1, 96.8) 95 68 (71.6) (61.4, 80.4) 60 3(5.0) (1.0, 13.9) 12 to <18 Months 9 8 (88.9) (51.8, 99.7) 45 32 (71.1)(55.7, 83.6) 31 1 (3.2) (0.1, 16.7) 18 to <24 Months 11 9 (81.8) (48.2,97.7) 50 36 (72.0) (57.5, 83.8) 29 2 (6.9) (0.8, 22.8) PMB2707 (B44)Before Vaccination 1 12 to <24 Months 19 0 (0.0)  (0.0, 17.6) 95 1 (1.1)(0.0, 5.7) 54 0 (0.0) (0.0, 6.6) 12 to <18 Months 9 0 (0.0)  (0.0, 33.6)46 1 (2.2)  (0.1, 11.5) 24 0 (0.0)  (0.0, 14.2) 18 to <24 Months 10 0(0.0)  (0.0, 30.8) 49 0 (0.0) (0.0, 7.3) 30 0 (0.0)  (0.0, 11.6) 1 Monthafter Vaccination 2 12 to <24 Months 19 13 (68.4) (43.4, 87.4) 94 64(68.1) (57.7, 77.3) 52 0 (0.0) (0.0, 6.8) 12 to <18 Months 9 7 (77.8)(40.0, 97.2) 47 34 (72.3) (57.4, 84.4) 23 0 (0.0)  (0.0, 14.8) 18 to <24Months 10 6 (60.0) (26.2, 87.8) 47 30 (63.8) (48.5, 77.3) 29 0 (0.0) (0.0, 11.9) 1 Month after Vaccination 3 12 to <24 Months 19 17 (89.5)(66.9, 98.7) 94 81 (86.2) (77.5, 92.4) 54 0 (0.0) (0.0, 6.6) 12 to <18Months 9 8 (88.9) (51.8, 99.7) 47 41 (87.2) (74.3, 95.2) 24 0 (0.0) (0.0, 14.2) 18 to <24 Months 10 9 (90.0) (55.5, 99.7) 47 40 (85.1)(71.7, 93.8) 30 0 (0.0)  (0.0, 11.6) Abbreviations: hSBA = serumbactericidal assay using human complement; LLOQ = lower limit ofquantitation. Note: LLOQ = 1:16 for A22; 1:8 for A56, B24, and B44.^(a)N = number of subjects with valid and determinate hSBA titers forthe given strain. ^(b)n = Number of subjects with observed hSBA titer≥LLOQ for the given strain at the given time point. ^(c)Exact 2-sided CIbased upon observed proportion of subjects, using the Clopper andPearson method.

hSBA GMTs

The hSBA GMTs for each of the 4 primary MnB test strains for subjects 12to <18 months of age, 18 to <24 months of age, and for the combined agestratum, at baseline, 1 month after the second vaccination, and 1 monthafter the third vaccination with bivalent rLP2086 was a secondaryendpoint. Table 26 provides hSBA GMTs for the 4 primary MnB strains forthe evaluable immunogenicity population.

For the combined age stratum, the hSBA GMTs at baseline were 8.0 and 8.4for PMB80 (A22); 4.0 and 4.1 for PMB2001 (A56); 4.4 and 4.1 for PMB2948(B24); and 4.0 and 4.0 for PMB2707 (B44) for the 60-μg and 120-μggroups, respectively.

At 1 month after Vaccination 2, the hSBA GMTs for the 12 to <18 monthsage stratum were 42.2 and 24.6 for PMB80 (A22); 101.6 and 117.2 forPMB2001 (A56); 10.6 and 6.0 for PMB2948 (B24); and 23.5 and 22.1 forPMB2707 (B44) for the 60-μg and 120-μg groups, respectively. For the 18to <24 months age stratum, the hSBA GMTs were 23.5 and 36.8 for PMB80(A22); 68.6 and 104.6 for PMB2001 (A56); 6.9 and 8.5 for PMB2948 (B24);and 21.1 and 17.0 for PMB2707 (B44) for the 60-μg and 120-μg groups,respectively. Overall, at 1 month after Vaccination 2 the hSBA GMTs forthe combined age stratum were 32.0 and 30.4 for PMB80 (A22); 82.6 and110.6 for PMB2001 (A56); 8.6 and 7.2 for PMB2948 (B24); and 22.2 and19.4 for PMB2707 (B44) for the 60-μg and 120-μg groups, respectively.

At 1 month after Vaccination 3, the hSBA GMTs for the 12 to <18 monthsage stratum were 80.6 and 63.0 for PMB80 (A22); 109.7 and 190.6 forPMB2001 (A56); 20.2 and 15.8 for PMB2948 (B24); and 29.6 and 46.3 forPMB2707 (B44) for the 60-μg and 120-μg groups, respectively. For the 18to <24 months age stratum, the hSBA GMTs were 82.3 and 71.4 for PMB80(A22); 181.0 and 154.4 for PMB2001 (A56); 17.0 and 14.5 for PMB2948(B24); and 34.3 and 44.9 for PMB2707 (B44) for the 60-μg and 120-μggroups, respectively. Overall, at 1 month after Vaccination 3, the hSBAGMTs for the combined age stratum were 81.6 and 67.3 for PMB80 (A22);142.8 and 171.4 for PMB2001 (A56); 18.4 and 15.1 for PMB2948 (B24); and32.0 and 45.6 for PMB2707 (B44) for the 60-μg and 120-μg groups,respectively.

In general, the hSBA GMTs for subjects in the HAV/saline group did notchange over time compared to baseline.

TABLE 26 hSBA GMTs for Primary Strains—Evaluable ImmunogenicityPopulation Strain (Variant) Vaccine Group (as Randomized) Sampling TimePoint 60 μg rLP2086 120 μg rLP2086 HAV/Saline Age Strata N^(a) GMT^(b)(95% CI)^(c) N^(a) GMT^(b) (95% CI)^(c) N^(a) GMT^(b) (95% CI)^(c) PMB80(A22) Before Vaccination 1 12 to <24 Months 20 8.0 (NE, NE) 97 8.4 (7.9,9.0) 61 8.1 (7.9, 8.3) 12 to <18 Months 9 8.0 (NE, NE) 46 8.5 (7.5, 9.6)31 8.0 (NE, NE) 18 to <24 Months 11 8.0 (NE, NE) 51 8.3 (7.8, 8.9) 308.2 (7.8, 8.6) 1 Month after Vaccination 2 12 to <24 Months 19 32.0(19.7, 52.0) 95 30.4 (24.3, 38.1) 59 8.3 (7.7, 8.9) 12 to <18 Months 1042.2 (22.6, 79.1) 45 24.6 (17.8, 34.2) 30 8.0 (NE, NE) 18 to <24 Months9 23.5 (10.1, 54.9) 50 36.8 (26.9, 50.3) 29 8.6  (7.4, 10.0) 1 Monthafter Vaccination 3 12 to <24 Months 20 81.6  (46.6, 142.8) 96 67.3(53.7, 84.3) 60 8.6 (7.9, 9.3) 12 to <18 Months 9 80.6  (30.9, 210.7) 4563.0 (44.5, 89.3) 31 8.6 (7.5, 9.8) 18 to <24 Months 11 82.3  (36.5,185.8) 51 71.4 (52.7, 96.6) 29 8.6 (7.7, 9.6) PMB2001 (A56) BeforeVaccination 1 12 to <24 Months 19 4.0 (NE, NE) 95 4.1 (3.9, 4.3) 53 4.0(NE, NE) 12 to <18 Months 9 4.0 (NE, NE) 46 4.0 (NE, NE) 24 4.0 (NE, NE)18 to <24 Months 10 4.0 (NE, NE) 49 4.2 (3.8, 4.5) 29 4.0 (NE, NE) 1Month after Vaccination 2 12 to <24 Months 19 82.6  (51.4, 132.9) 95110.6  (92.0, 133.0) 52 4.0 (NE, NE) 12 to <18 Months 9 101.6  (64.0,161.2) 47 117.2  (89.7, 153.0) 23 4.0 (NE, NE) 18 to <24 Months 10 68.6 (28.2, 166.8) 48 104.6  (80.4, 136.0) 29 4.0 (NE, NE) 1 Month afterVaccination 3 12 to <24 Months 19 142.8  (85.5, 238.6) 95 171.4 (141.6,207.4) 54 4.2 (3.8, 4.5) 12 to <18 Months 9 109.7  (70.4, 171.1) 47190.6 (146.9, 247.4) 24 4.0 (NE, NE) 18 to <24 Months 10 181.0  (68.6,477.9) 48 154.4 (116.3, 205.1) 30 4.3 (3.7, 4.9) PMB2948 (B24) BeforeVaccination 1 12 to <24 Months 21 4.4 (3.6, 5.4) 97 4.1 (4.0, 4.3) 614.2 (3.8, 4.6) 12 to <18 Months 10 4.0 (NE, NE) 46 4.1 (3.9, 4.4) 31 4.0(NE, NE) 18 to <24 Months 11 4.8 (3.2, 7.4) 51 4.1 (3.9, 4.3) 30 4.4(3.6, 5.3) 1 Month after Vaccination 2 12 to <24 Months 19 8.6  (6.1,12.2) 86 7.2 (5.9, 8.7) 59 4.1 (3.9, 4.4) 12 to <18 Months 10 10.6 (6.2, 18.0) 42 6.0 (4.7, 7.8) 30 4.0 (NE, NE) 18 to <24 Months 9 6.9 (4.1, 11.5) 44 8.5  (6.4, 11.3) 29 4.3 (3.7, 5.0) 1 Month afterVaccination 3 12 to <24 Months 20 18.4 (11.8, 28.6) 95 15.1 (12.3, 18.6)60 4.3 (3.9, 4.8) 12 to <18 Months 9 20.2 (11.1, 36.6) 45 15.8 (11.4,21.8) 31 4.3 (3.7, 4.9) 18 to <24 Months 11 17.0  (8.2, 35.5) 50 14.5(11.1, 19.1) 29 4.4 (3.8, 5.0) PMB2707 (B44) Before Vaccination 1 12 to<24 Months 19 4.0 (NE, NE) 95 4.0 (4.0, 4.1) 54 4.0 (NE, NE) 12 to <18Months 9 4.0 (NE, NE) 46 4.1 (3.9, 4.2) 24 4.0 (NE, NE) 18 to <24 Months10 4.0 (NE, NE) 49 4.0 (NE, NE) 30 4.0 (NE, NE) 1 Month afterVaccination 2 12 to <24 Months 19 22.2 (11.2, 43.9) 94 19.4 (15.1, 24.9)52 4.0 (NE, NE) 12 to <18 Months 9 23.5  (9.3, 59.4) 47 22.1 (15.5,31.6) 23 4.0 (NE, NE) 18 to <24 Months 10 21.1  (6.5, 68.3) 47 17.0(11.8, 24.4) 29 4.0 (NE, NE) 1 Month after Vaccination 3 12 to <24Months 19 32.0 (18.3, 55.8) 94 45.6 (35.2, 59.0) 54 4.0 (NE, NE) 12 to<18 Months 9 29.6 (11.6, 75.8) 47 46.3 (31.6, 67.8) 24 4.0 (NE, NE) 18to <24 Months 10 34.3 (15.0, 78.2) 47 44.9 (31.3, 64.5) 30 4.0 (NE, NE)Abbreviations: GMT = geometric mean titer; hSBA = serum bactericidalassay using human complement; LLOQ = lower limit of quantitation; NE =not estimable Note: LLOQ = 1:16 for A22; 1:8 for A56, B24, and B44.Titers below the LLOQ were set to 0.5 × LLOQ for analysis. ^(a)N =number of subjects with valid and determinate hSBA titers for the givenstrain. ^(b)GMTs were calculated using all subjects with valid anddeterminate hSBA titers at the given time point. ^(c)CIs are backtransformations of confidence levels based on the Student t distributionfor the mean logarithm of the hSBA titers.

Results for the mITT population were similar to those of the evaluableimmunogenicity population.

Subgroup analyses of hSBA GMTs for each of the 4 primary MnB teststrains were assessed for the evaluable immunogenicity population by sexand country. There were no clinically important differences observed inthe subgroup analyses performed.

Defined hSBA Titers

The proportions of subjects 12 to <18 months of age, 18 to <24 months ofage, and for the combined age stratum, achieving hSBA titers ≥1:4, ≥1:8,≥1:16, ≥1:32, ≥1:64, and ≥1:128 for each of the 4 primary MnB teststrains at baseline, 1 month after the second vaccination, and 1 monthafter the third vaccination with bivalent rLP2086 was a secondaryimmunogenicity endpoint.

The proportion of subjects achieving defined hSBA titers for the 4primary MnB strains is presented in Table 27 for the evaluableimmunogenicity population.

The results for subjects receiving bivalent rLP2086 who achieved an hSBAtiter ≥1:4 and ≥1:16 are described below. An hSBA titer of ≥1:4 iswidely recognized as the correlate of protection against IMD; however, amore conservative hSBA titer of ≥1:16 has been considered a levelindicative of a 4-fold vaccine effect for subjects seronegative beforevaccination.

In general, the proportion of subjects in the HAV/saline group achievingdefined hSBA titers did not change overtime compared to baseline.

Results for the mITT population were similar to those of the evaluableimmunogenicity population.

hSBA Titer ≥1:4

At 1 month after Vaccination 2, the proportion of subjects 12 to <18months of age achieving an hSBA titer ≥1:4 was 90.0% and 64.4% for PMB80(A22); 100.0% and 100.0% for PMB2001 (A56); 70.0% and 28.6% for PMB2948(B24); and 77.8% and 72.3% for PMB2707 (B44) for the 60-μg and 120-μggroups, respectively. The proportion of subjects 18 to <24 months of ageachieving an hSBA titer ≥1:4 was 66.7% and 86.0% for PMB80 (A22); 100.0%and 100.0% for PMB2001 (A56); 44.4% and 43.2% for PMB2948 (B24); and70.0% and 63.8% for PMB2707 (B44) for the 60-μg and 120-μg groups,respectively. Overall for the combined age stratum, the proportion ofsubjects achieving an hSBA titer ≥1:4 at 1 month after Vaccination 2 was78.9% and 75.8% for PMB80 (A22); 100.0% and 100.0% for PMB2001 (A56);57.9% and 36.0% for PMB2948 (B24); and 73.7% and 68.1% for PMB2707 (B44)for the 60-μg and 120-μg groups, respectively.

At 1 month after Vaccination 3, the proportion of subjects 12 to <18months of age achieving an hSBA titer ≥1:4 was 88.9% and 91.1% for PMB80(A22); 100.0% and 100.0% for PMB2001 (A56); 88.9% and 71.1% for PMB2948(B24); and 88.9% and 87.2% for PMB2707 (B44) for the 60-μg and 120-μggroups, respectively. The proportion of subjects 18 to <24 months of ageachieving an hSBA titer ≥1:4 was 90.9% and 88.2% for PMB80 (A22); 100.0%and 100.0% for PMB2001 (A56); 81.8% and 72.0% for PMB2948 (B24); and90.0% and 87.2% for PMB2707 (B44) for the 60-μg and 120-μg groups,respectively. Overall for the combined age stratum, the proportion ofsubjects achieving an hSBA titer ≥1:4 at 1 month after Vaccination 3 was90.0% and 89.6% for PMB80 (A22); 100.0% and 100.0% for PMB2001 (A56);85.0% and 71.6% for PMB2948 (B24); and 89.5% and 87.2% for PMB2707 (B44)for the 60-μg and 120-μg groups, respectively.

hSBA Titer ≥1:16

At 1 month after Vaccination 2, the proportion of subjects 12 to <18months of age achieving an hSBA titer ≥1:16 was 90.0% and 64.4% forPMB80 (A22); 100.0% and 100.0% for PMB2001 (A56); 60.0% and 21.4% forPMB2948 (B24); and 77.8% and 72.3% for PMB2707 (B44) for the 60-μg and120-μg groups, respectively. The proportion of subjects 18 to <24 monthsof age achieving an hSBA titer ≥1:16 was 66.7% and 86.0% for PMB80(A22); 90.0% and 100.0% for PMB2001 (A56); 33.3% and 43.2% for PMB2948(B24); and 60.0% and 61.7% for PMB2707 (B44) for the 60-μg and 120-μggroups, respectively. Overall for the combined age stratum, theproportion of subjects achieving an hSBA titer ≥1:16 at 1 month afterVaccination 2 was 78.9% and 74.7% for PMB80 (A22); 94.7% and 100.0% forPMB2001 (A56); 47.4% and 32.6% for PMB2948 (B24); and 68.4% and 67.0%for PMB2707 (B44) for the 60-μg and 120-μg groups, respectively.

At 1 month after Vaccination 3, the proportion of subjects 12 to <18months of age achieving an hSBA titer ≥1:16 was 88.9% and 91.1% forPMB80 (A22); 100.0% and 97.9% for PMB2001 (A56); 88.9% and 66.7% forPMB2948 (B24); and 77.8% and 87.2% for PMB2707 (B44) for the 60-μg and120-μg groups, respectively. The proportion of subjects 18 to <24 monthsof age achieving an hSBA titer ≥1:16 was 90.9% and 88.2% for PMB80(A22); 100.0% and 100.0% for PMB2001 (A56); 63.6% and 68.0% for PMB2948(B24); and 90.0% and 85.1% for PMB2707 (B44) for the 60-μg and 120-μggroups, respectively. Overall for the combined age stratum, theproportion of subjects achieving an hSBA titer ≥1:16 at 1 month afterVaccination 3 was 90.0% and 89.6% for PMB80 (A22); 100.0% and 98.9% forPMB2001 (A56); 75.0% and 67.4% for PMB2948 (B24); and 84.2% and 86.2%for PMB2707 (B44) for the 60-μg and 120-μg groups, respectively.

TABLE 27 Subjects Achieving Defined hSBA Titers for Primary Strains -Evaluable Immunogenicity Population Strain (Variant) Vaccine Group (asRandomized) Sampling Time Point 60 μg rLP2086 120 μg rLP2086 HAV/SalineAge Strata Titer N^(a) n^(b) % (95% CI)^(c) N^(a) n^(b) % (95% CI)^(c)N^(a) n^(b) % (95% CI)^(c) PMB80 (A22) Before Vaccination 1 12 to <24Months 4 20 0 0.0  (0.0, 16.8) 97 4 4.1  (1.1, 10.2) 61 1 1.6 (0.0, 8.8)8 20 0 0.0  (0.0, 16.8) 97 3 3.1 (0.6, 8.8) 61 1 1.6 (0.0, 8.8) 16 20 00.0  (0.0, 16.8) 97 3 3.1 (0.6, 8.8) 61 1 1.6 (0.0, 8.8) 32 20 0 0.0 (0.0, 16.8) 97 2 2.1 (0.3, 7.3) 61 0 0.0 (0.0, 5.9) 64 20 0 0.0  (0.0,16.8) 97 1 1.0 (0.0, 5.6) 61 0 0.0 (0.0, 5.9) 128 20 0 0.0  (0.0, 16.8)97 1 1.0 (0.0, 5.6) 61 0 0.0 (0.0, 5.9) 12 to <18 Months 4 9 0 0.0 (0.0, 33.6) 46 2 4.3  (0.5, 14.8) 31 0 0.0  (0.0, 11.2) 8 9 0 0.0 (0.0, 33.6) 46 1 2.2  (0.1, 11.5) 31 0 0.0  (0.0, 11.2) 16 9 0 0.0 (0.0, 33.6) 46 1 2.2  (0.1, 11.5) 31 0 0.0  (0.0, 11.2) 32 9 0 0.0 (0.0, 33.6) 46 1 2.2  (0.1, 11.5) 31 0 0.0  (0.0, 11.2) 64 9 0 0.0 (0.0, 33.6) 46 1 2.2  (0.1, 11.5) 31 0 0.0  (0.0, 11.2) 128 9 0 0.0 (0.0, 33.6) 46 1 2.2  (0.1, 11.5) 31 0 0.0  (0.0, 11.2) 18 to <24Months 4 11 0 0.0  (0.0, 28.5) 51 2 3.9  (0.5, 13.5) 30 1 3.3  (0.1,17.2) 8 11 0 0.0  (0.0, 28.5) 51 2 3.9  (0.5, 13.5) 30 1 3.3  (0.1,17.2) 16 11 0 0.0  (0.0, 28.5) 51 2 3.9  (0.5, 13.5) 30 1 3.3  (0.1,17.2) 32 11 0 0.0  (0.0, 28.5) 51 1 2.0  (0.0, 10.4) 30 0 0.0  (0.0,11.6) 64 11 0 0.0  (0.0, 28.5) 51 0 0.0 (0.0, 7.0) 30 0 0.0  (0.0, 11.6)128 11 0 0.0  (0.0, 28.5) 51 0 0.0 (0.0, 7.0) 30 0 0.0  (0.0, 11.6) 1Month after Vaccination 2 12 to <24 Months 4 19 15 78.9 (54.4, 93.9) 9572 75.8 (65.9, 84.0) 59 1 1.7 (0.0, 9.1) 8 19 15 78.9 (54.4, 93.9) 95 7275.8 (65.9, 84.0) 59 1 1.7 (0.0, 9.1) 16 19 15 78.9 (54.4, 93.9) 95 7174.7 (64.8, 83.1) 59 1 1.7 (0.0, 9.1) 32 19 12 63.2 (38.4, 83.7) 95 5658.9 (48.4, 68.9) 59 1 1.7 (0.0, 9.1) 64 19 7 36.8 (16.3, 61.6) 95 3334.7 (25.3, 45.2) 59 1 1.7 (0.0, 9.1) 128 19 4 21.1  (6.1, 45.6) 95 1313.7  (7.5, 22.3) 59 0 0.0 (0.0, 6.1) 12 to <18 Months 4 10 9 90.0(55.5, 99.7) 45 29 64.4 (48.8, 78.1) 30 0 0.0  (0.0, 11.6) 8 10 9 90.0(55.5, 99.7) 45 29 64.4 (48.8, 78.1) 30 0 0.0  (0.0, 11.6) 16 10 9 90.0(55.5, 99.7) 45 29 64.4 (48.8, 78.1) 30 0 0.0  (0.0, 11.6) 32 10 8 80.0(44.4, 97.5) 45 23 51.1 (35.8, 66.3) 30 0 0.0  (0.0, 11.6) 64 10 5 50.0(18.7, 81.3) 45 13 28.9 (16.4, 44.3) 30 0 0.0  (0.0, 11.6) 128 10 2 20.0 (2.5, 55.6) 45 5 11.1  (3.7, 24.1) 30 0 0.0  (0.0, 11.6) 18 to <24Months 4 9 6 66.7 (29.9, 92.5) 50 43 86.0 (73.3, 94.2) 29 1 3.4  (0.1,17.8) 8 9 6 66.7 (29.9, 92.5) 50 43 86.0 (73.3, 94.2) 29 1 3.4  (0.1,17.8) 16 9 6 66.7 (29.9, 92.5) 50 42 84.0 (70.9, 92.8) 29 1 3.1  (0.1,17.8) 32 9 4 44.4 (13.7, 78.8) 50 33 66.0 (51.2, 78.8) 29 1 3.4  (0.1,17.8) 64 9 2 22.2  (2.8, 60.0) 50 20 40.0 (26.4, 54.8) 29 1 3.4  (0.1,17.8) 128 9 2 22.2  (2.8, 60.0) 50 8 16.0  (7.2, 29.1) 29 0 0.0  (0.0,11.9) 1 Month after Vaccination 3 12 to <24 Months 4 20 18 90.0 (68.3,98.8) 96 86 89.6 (81.7, 94.9) 60 4 6.7  (1.8, 16.2) 8 20 18 90.0 (68.3,98.8) 96 86 89.6 (81.7, 94.9) 60 4 6.7  (1.8, 16.2) 16 20 18 90.0 (68.3,98.8) 96 86 89.6 (81.7, 94.9) 60 3 5.0  (1.0, 13.9) 32 20 17 85.0 (62.1,96.8) 96 81 84.4 (75.5, 91.0) 60 2 3.3  (0.4, 11.5) 64 20 14 70.0 (45.7,88.1) 96 64 66.7 (56.3, 76.0) 60 1 1.7 (0.0, 8.9) 128 20 10 50.0 (27.2,72.8) 96 42 43.8 (33.6, 54.3) 60 0 0.0 (0.0, 6.0) 12 to <18 Months 4 9 888.9 (51.8, 99.7) 45 41 91.1 (78.8, 97.5) 31 2 6.5  (0.8, 21.4) 8 9 888.9 (51.8, 99.7) 45 41 91.1 (78.8, 97.5) 31 2 6.5  (0.8, 21.4) 16 9 888.9 (51.8, 99.7) 45 41 91.1 (78.8, 97.5) 31 1 3.2  (0.1, 16.7) 32 9 888.9 (51.8, 99.7) 45 37 82.2 (67.9, 92.0) 31 1 3.2  (0.1, 16.7) 64 9 666.7 (29.9, 92.5) 45 27 60.0 (44.3, 74.3) 31 1 3.2  (0.1, 16.7) 128 9 444.4 (13.7, 78.8) 45 17 37.8 (23.8, 53.5) 31 0 0.0  (0.0, 11.2) 18 to<24 Months 4 11 10 90.9 (58.7, 99.8) 51 45 88.2 (76.1, 95.6) 29 2 6.9 (0.8, 22.8) 8 11 10 90.9 (58.7, 99.8) 51 45 88.2 (76.1, 95.6) 29 2 6.9 (0.8, 22.8) 16 11 10 90.9 (58.7, 99.8) 51 45 88.2 (76.1, 95.6) 29 2 6.9 (0.8, 22.8) 32 11 9 81.8 (48.2, 97.7) 51 44 86.3 (73.7, 94.3) 29 1 3.4 (0.1, 17.8) 64 11 8 72.7 (39.0, 94.0) 51 37 72.5 (58.3, 84.1) 29 0 0.0 (0.0, 11.9) 128 11 6 54.5 (23.4, 83.3) 51 25 49.0 (34.8, 63.4) 29 0 0.0 (0.0, 11.9) PMB2001 (A56) Before Vaccination 1 12 to <24 Months 4 19 00.0  (0.0, 17.6) 95 2 2.1 (0.3, 7.4) 53 1 1.9  (0.0, 10.1) 8 19 0 0.0 (0.0, 17.6) 95 1 1.1 (0.0, 5.7) 53 0 0.0 (0.0, 6.7) 16 19 0 0.0  (0.0,17.6) 95 1 1.1 (0.0, 5.7) 53 0 0.0 (0.0, 6.7) 32 19 0 0.0  (0.0, 17.6)95 1 1.1 (0.0, 5.7) 53 0 0.0 (0.0, 6.7) 64 19 0 0.0  (0.0, 17.6) 95 00.0 (0.0, 3.8) 53 0 0.0 (0.0, 6.7) 128 19 0 0.0  (0.0, 17.6) 95 0 0.0(0.0, 3.8) 53 0 0.0 (0.0, 6.7) 12 to <18 Months 4 9 0 0.0  (0.0, 33.6)46 0 0.0 (0.0, 7.7) 24 1 4.2  (0.1, 21.1) 8 9 0 0.0  (0.0, 33.6) 46 00.0 (0.0, 7.7) 24 0 0.0  (0.0, 14.2) 16 9 0 0.0  (0.0, 33.6) 46 0 0.0(0.0, 7.7) 24 0 0.0  (0.0, 14.2) 32 9 0 0.0  (0.0, 33.6) 46 0 0.0 (0.0,7.7) 24 0 0.0  (0.0, 14.2) 64 9 0 0.0  (0.0, 33.6) 46 0 0.0 (0.0, 7.7)24 0 0.0  (0.0, 14.2) 128 9 0 0.0  (0.0, 33.6) 46 0 0.0 (0.0, 7.7) 24 00.0  (0.0, 14.2) 18 to <24 Months 4 10 0 0.0  (0.0, 30.8) 49 2 4.1 (0.5, 14.0) 29 0 0.0  (0.0, 11.9) 8 10 0 0.0  (0.0, 30.8) 49 1 2.0 (0.1, 10.9) 29 0 0.0  (0.0, 11.9) 16 10 0 0.0  (0.0, 30.8) 49 1 2.0 (0.1, 10.9) 29 0 0.0  (0.0, 11.9) 32 10 0 0.0  (0.0, 30.8) 49 1 2.0 (0.1, 10.9) 29 0 0.0  (0.0, 11.9) 64 10 0 0.0  (0.0, 30.8) 49 0 0.0(0.0, 7.3) 29 0 0.0  (0.0, 11.9) 128 10 0 0.0  (0.0, 30.8) 49 0 0.0(0.0, 7.3) 29 0 0.0  (0.0, 11.9) 8 10 0 0.0  (0.0, 30.8) 49 1 2.0  (0.1,10.9) 29 0 0.0  (0.0, 11.9) 16 10 0 0.0  (0.0, 30.8) 49 1 2.0  (0.1,10.9) 29 0 0.0  (0.0, 11.9) 32 10 0 0.0  (0.0, 30.8) 49 1 2.0  (0.1,10.9) 29 0 0.0  (0.0, 11.9) 64 10 0 0.0  (0.0, 30.8) 49 0 0.0 (0.0, 7.3)29 0 0.0  (0.0, 11.9) 128 10 0 0.0  (0.0, 30.8) 49 0 0.0 (0.0, 7.3) 29 00.0  (0.0, 11.9) 1 Month after Vaccination 2 12 to <24 Months 4 19 19100.0  (82.4, 100.0) 95 95 100.0  (96.2, 100.0) 52 1 1.9  (0.0, 10.3) 819 18 94.7 (74.0, 99.9) 95 95 100.0  (96.2, 100.0) 52 0 0.0 (0.0, 6.8)16 19 18 94.7 (74.0, 99.9) 95 95 100.0  (96.2, 100.0) 52 0 0.0 (0.0,6.8) 32 19 18 94.7 (74.0, 99.9) 95 91 95.8 (89.6, 98.8) 52 0 0.0 (0.0,6.8) 64 19 16 84.2 (60.4, 96.6) 95 82 86.3 (77.7, 92.5) 52 0 0.0(0.0,6.8) 128 19 9 47.4 (24.4, 71.1) 95 54 56.8 (46.3, 67.0) 52 0 0.0(0.0, 6.8) 12 to <18 Months 4 9 9 100.0  (66.4, 100.0) 47 47 100.0 (92.5, 100.0) 23 1 4.3  (0.1, 21.9) 8 9 9 100.0  (66.4, 100.0) 47 47100.0  (92.5, 100.0) 23 0 0.0  (0.0, 14.8) 16 9 9 100.0  (66.4, 100.0)47 47 100.0  (92.5, 100.0) 23 0 0.0  (0.0, 14.8) 32 9 9 100.0  (66.4.100.0) 47 45 95.7 (85.5, 99.5) 23 0 0.0  (0.0. 14.8) 64 9 9 100.0 (66.4, 100.0) 47 41 87.2 (74.3, 95.2) 23 0 0.0  (0.0, 14.8) 128 9 444.4 (13.7, 78.8) 47 28 59.6 (44.3, 73.6) 23 0 0.0  (0.0, 14.8) 18 to<24 Months 4 10 10 100.0  (69.2, 100.0) 48 48 100.0  (92.6, 100.0) 29 00.0  (0.0, 11.9) 8 10 9 90.0 (55.5, 99.7) 48 48 100.0  (92.6, 100.0) 290 0.0  (0.0, 11.9) 16 10 9 90.0 (55.5, 99.7) 48 48 100.0  (92.6, 100.0)29 0 0.0  (0.0, 11.9) 32 10 9 90.0 (55.5, 99.7) 48 46 95.8 (85.7, 99.5)29 0 0.0  (0.0, 11.9) 64 10 7 70.0 (34.8, 93.3) 48 41 85.4 (72.2, 93.9)29 0 0.0  (0.0, 11.9) 128 10 5 50.0 (18.7, 81.3) 48 26 54.2 (39.2, 68.6)29 0 0.0  (0.0, 11.9) 1 Month after Vaccination 3 12 to <24 Months 4 1919 100.0  (82.4, 100.0) 95 95 100.0  (96.2, 100.0) 54 5 9.3  (3.1, 20.3)8 19 19 100.0  (82.4, 100.0) 95 95 100.0  (96.2, 100.0) 54 1 1.9 (0.0,9.9) 16 19 19 100.0  (82.4, 100.0) 95 94 98.9  (94.3, 100.0) 54 1 1.9(0.0, 9.9) 32 19 18 94.7 (74.0, 99.9) 95 91 95.8 (89.6, 98.8) 54 1 1.9(0.0, 9.9) 64 19 17 89.5 (66.9, 98.7) 95 85 89.5 (81.5, 94.8) 54 0 0.0(0.0, 6.6) 128 19 13 68.4 (43.4, 87.4) 95 79 83.2 (74.1, 90.1) 54 0 0.0(0.0, 6.6) 12 to <18 Months 4 9 9 100.0  (66.4, 100.0) 47 47 100.0 (92.5, 100.0) 24 2 8.3  (1.0, 27.0) 8 9 9 100.0  (66.4, 100.0) 47 47100.0  (92.5, 100.0) 24 0 0.0  (0.0, 14.2) 16 9 9 100.0  (66.4, 100.0)47 46 97.9 (88.7, 99.9) 24 0 0.0  (0.0, 14.2) 32 9 9 100.0  (66.4,100.0) 47 46 97.9 (88.7, 99.9) 24 0 0.0  (0.0, 14.2) 64 9 9 100.0 (66.4, 100.0) 47 44 93.6 (82.5, 98.7) 24 0 0.0  (0.0, 14.2) 128 9 555.6 (21.2, 86.3) 47 42 89.4 (76.9, 96.5) 24 0 0.0  (0.0, 14.2) 18 to<24 Months 4 10 10 100.0  (69.2, 100.0) 48 48 100.0  (92.6, 100.0) 30 310.0  (2.1, 26.5) 8 10 10 100.0  (69.2, 100.0) 48 48 100.0  (92.6,100.0) 30 1 3.3  (0.1, 17.2) 16 10 10 100.0  (69.2, 100.0) 48 48 100.0 (92.6, 100.0) 30 1 3.3  (0.1, 17.2) 32 10 9 90.0 (55.5, 99.7) 48 4593.8 (82.8, 98.7) 30 1 3.3  (0.1, 17.2) 64 10 8 80.0 (44.4, 97.5) 48 4185.4 (72.2, 93.9) 30 0 0.0  (0.0, 11.6) 128 10 8 80.0 (44.4, 97.5) 48 3777.1 (62.7, 88.0) 30 0 0.0  (0.0, 11.6) PMB2948 (B24) Before Vaccination1 12 to <24 Months 4 21 1 4.8  (0.1, 23.8) 97 2 2.1 (0.3, 7.3) 61 1 1.6(0.0, 8.8) 8 21 1 4.8  (0.1, 23.8) 97 2 2.1 (0.3, 7.3) 61 1 1.6 (0.0,8.8) 16 21 1 4.8  (0.1, 23.8) 97 2 2.1 (0.3, 7.3) 61 1 1.6 (0.0, 8.8) 3221 1 4.8  (0.1, 23.8) 97 0 0.0 (0.0, 3.7) 61 1 1.6 (0.0, 8.8) 64 21 00.0  (0.0, 16.1) 97 0 0.0 (0.0, 3.7) 61 1 1.6 (0.0, 8.8) 128 21 0 0.0 (0.0, 16.1) 97 0 0.0 (0.0, 3.7) 61 0 0.0 (0.0, 5.9) 12 to <18 Months 410 0 0.0  (0.0, 30.8) 46 1 2.2  (0.1, 11.5) 31 0 0.0  (0.0, 11.2) 8 10 00.0  (0.0, 30.8) 46 1 2.2  (0.1, 11.5) 31 0 0.0  (0.0, 11.2) 16 10 0 0.0 (0.0, 30.8) 46 1 2.2  (0.1, 11.5) 31 0 0.0  (0.0, 11.2) 32 10 0 0.0 (0.0, 30.8) 46 0 0.0 (0.0, 7.7) 31 0 0.0  (0.0, 11.2) 64 10 0 0.0 (0.0, 30.8) 46 0 0.0 (0.0, 7.7) 31 0 0.0  (0.0, 11.2) 128 10 0 0.0 (0.0, 30.8) 46 0 0.0 (0.0, 7.7) 31 0 0.0  (0.0, 11.2) 18 to <24 Months4 11 1 9.1  (0.2, 41.3) 51 1 2.0  (0.0, 10.4) 30 1 3.3  (0.0, 17.2) 8 111 9.1  (0.2, 41.3) 51 1 2.0  (0.0, 10.4) 30 1 3.3  (0.0, 17.2) 16 11 19.1  (0.2, 41.3) 51 1 2.0  (0.0, 10.4) 30 1 3.3  (0.0, 17.2) 32 11 1 9.1 (0.2, 41.3) 51 1 0.0 (0.0, 7.0) 30 1 3.3  (0.0, 17.2) 64 11 0 0.0 (0.0, 28.5) 51 1 0.0 (0.0, 7.0) 30 1 3.3  (0.0, 17.2) 128 11 0 0.0 (0.0, 28.5) 51 1 0.0 (0.0, 7.0) 30 0 0.0  (0.0, 11.6) 1 Month afterVaccination 2 12 to <24 Months 4 19 11 57.9 (33.5, 79.7) 86 31 36.3(26.0, 47.1) 59 1 1.7 (0.0, 9.1) 8 19 11 57.9 (33.5, 79.7) 86 29 33.7(23.9, 44.7) 59 1 1.7 (0.0, 9.1) 16 19 9 47.4 (24.4, 71.1) 86 28 32.6(22.8, 43.5) 59 1 1.7 (0.0, 9.1) 32 19 1 5.3  (0.1, 26.0) 86 12 14.0 (7.4, 23.1) 59 1 1.7 (0.0, 9.1) 64 19 0 0.0  (0.0, 17.6) 86 3 3.5 (0.7,9.9) 59 0 0.0 (0.0, 6.1) 128 19 0 0.0  (0.0, 17.6) 86 1 1.2 (0.0, 6.3)59 0 0.0 (0.0, 6.1) 12 to <18 Months 4 10 7 70.0 (34.8, 93.3) 42 12 28.6(15.7, 44.6) 30 0 0.0  (0.0, 11.6) 8 10 7 70.0 (34.8, 93.3) 42 10 23.8(12.1, 39.5) 30 0 0.0  (0.0, 11.6) 16 10 6 60.0 (26.2, 87.8) 42 9 21.4(10.3, 36.8) 30 0 0.0  (0.0, 11.6) 32 10 1 10.0  (0.3, 44.5) 42 4 9.5 (2.7, 22.6) 30 0 0.0  (0.0, 11.6) 64 10 0 0.0  (0.0, 30.8) 42 1 2.4 (0.1, 12.6) 30 0 0.0  (0.0, 11.6) 128 10 0 0.0  (0.0, 30.8) 42 1 2.4 (0.1, 12.6) 30 0 0.0  (0.0, 11.6) 18 to <24 Months 4 9 4 44.4 (13.7,78.8) 44 19 43.2 (28.3, 59.0) 29 1 3.4  (0.1, 17.8) 8 9 4 44.4 (13.7,78.8) 44 19 43.2 (28.3, 59.0) 29 1 3.4  (0.1, 17.8) 16 9 3 33.3  (7.5,70.1) 44 19 43.2 (28.3, 59.0) 29 1 3.4  (0.1, 17.8) 32 9 0 0.0  (0.0,33.6) 44 8 18.2  (8.2, 32.7) 29 1 3.4  (0.1, 17.8) 64 9 0 0.0  (0.0,33.6) 44 2 4.5  (0.6, 15.5) 29 0 0.0  (0.0, 11.9) 128 9 0 0.0  (0.0,33.6) 44 0 0.0 (0.0, 8.0) 29 0 0.0  (0.0, 11.9) 1 Month afterVaccination 3 12 to <24 Months 4 20 17 85.0 (62.1, 96.8) 95 68 71.6(61.4, 80.4) 60 3 5.0  (1.0, 13.9) 8 20 17 85.0 (62.1, 96.8) 95 68 71.6(61.4, 80.4) 60 3 5.0  (1.0, 13.9) 16 20 15 75.0 (50.9, 91.3) 95 64 67.4(57.0, 76.6) 60 3 5.0  (1.0, 13.9) 32 20 8 40.0 (19.1, 63.9) 95 34 35.8(26.2, 46.3) 60 1 1.7 (0.0, 8.9) 64 20 3 15.0  (3.2, 37.9) 95 13 13.7 (7.5, 22.3) 60 0 0.0 (0.0, 6.0) 128 20 1 5.0  (0.1, 24.9) 95 2 2.1(0.3, 7.4) 60 0 0.0 (0.0, 6.0) 12 to <18 Months 4 9 8 88.9 (51.8, 99.7)45 32 71.1 (55.7, 83.6) 31 1 3.2  (0.1, 16.7) 8 9 8 88.9 (51.8, 99.7) 4532 71.1 (55.7, 83.6) 31 1 3.2  (0.1, 16.7) 16 9 8 88.9 (51.8, 99.7) 4530 66.7 (51.0, 80.0) 31 1 3.2  (0.1, 16.7) 32 9 4 44.4 (13.7, 78.8) 4517 37.8 (23.8, 53.5) 31 1 3.2  (0.1, 16.7) 64 9 1 11.1  (0.3, 48.2) 45 817.8  (8.0, 32.1) 31 0 0.0  (0.0, 11.2) 128 9 0 0.0  (0.0, 33.6) 45 12.2  (0.1, 11.8) 31 0 0.0  (0.0, 11.2) 18 to <24 Months 4 11 9 81.8(48.2, 97.7) 50 36 72.0 (57.5, 83.8) 29 2 6.9  (0.8, 22.8) 8 11 9 81.8(48.2, 97.7) 50 36 72.0 (57.5, 83.8) 29 2 6.9  (0.8, 22.8) 16 11 7 63.6(30.8, 89.1) 50 34 68.0 (53.3, 80.5) 29 2 6.9  (0.8, 22.8) 32 11 4 36.4(10.9, 69.2) 50 17 34.0 (21.2, 48.8) 29 0 0.0  (0.0, 11.9) 64 11 2 18.2 (2.3, 51.8) 50 5 10.0  (3.3, 21.8) 29 0 0.0  (0.0, 11.9) 128 11 1 9.1 (0.2, 41.3) 50 1 2.0  (0.1, 10.6) 29 0 0.0  (0.0, 11.9) PMB2707 (B44)Before Vaccination 1 12 to <24 Months 4 19 0 0.0  (0.0, 17.6) 95 1 1.1(0.0, 5.7) 54 0 0.0 (0.0, 6.6) 8 19 0 0.0  (0.0, 17.6) 95 1 1.1 (0.0,5.7) 54 0 0.0 (0.0, 6.6) 16 19 0 0.0  (0.0, 17.6) 95 0 0.0 (0.0, 3.8) 540 0.0 (0.0, 6.6) 32 19 0 0.0  (0.0, 17.6) 95 0 0.0 (0.0, 3.8) 54 0 0.0(0.0, 6.6) 64 19 0 0.0  (0.0, 17.6) 95 0 0.0 (0.0, 3.8) 54 0 0.0 (0.0,6.6) 128 19 0 0.0  (0.0, 17.6) 95 0 0.0 (0.0, 3.8) 54 0 0.0 (0.0, 6.6)12 to <18 Months 4 9 0 0.0  (0.0, 33.6) 46 1 2.2  (0.1, 11.5) 24 0 0.0 (0.0, 14.2) 8 9 0 0.0  (0.0, 33.6) 46 1 2.2  (0.1, 11.5) 24 0 0.0 (0.0, 14.2) 16 9 0 0.0  (0.0, 33.6) 46 0 0.0 (0.0, 7.7) 24 0 0.0  (0.0,14.2) 32 9 0 0.0  (0.0, 33.6) 46 0 0.0 (0.0, 7.7) 24 0 0.0  (0.0, 14.2)64 9 0 0.0  (0.0, 33.6) 46 0 0.0 (0.0, 7.7) 24 0 0.0  (0.0, 14.2) 128 90 0.0  (0.0, 33.6) 46 0 0.0 (0.0, 7.7) 24 0 0.0  (0.0, 14.2) 18 to <24Months 4 10 0 0.0  (0.0, 30.8) 49 0 0.0 (0.0, 7.3) 30 0 0.0  (0.0, 11.6)8 10 0 0.0  (0.0, 30.8) 49 0 0.0 (0.0, 7.3) 30 0 0.0  (0.0, 11.6) 16 100 0.0  (0.0, 30.8) 49 0 0.0 (0.0, 7.3) 30 0 0.0  (0.0, 11.6) 32 10 0 0.0 (0.0, 30.8) 49 0 0.0 (0.0, 7.3) 30 0 0.0  (0.0, 11.6) 64 10 0 0.0 (0.0, 30.8) 49 0 0.0 (0.0, 7.3) 30 0 0.0  (0.0, 11.6) 128 10 0 0.0 (0.0, 30.8) 49 0 0.0 (0.0, 7.3) 30 0 0.0  (0.0, 11.6) 1 Month afterVaccination 2 12 to <24 Months 4 19 14 73.7 (48.8, 90.9) 94 64 68.1(57.7, 77.3) 52 0 0.0 (0.0, 6.8) 8 19 13 68.4 (43.4, 87.4) 94 64 68.1(57.7, 77.3) 52 0 0.0 (0.0, 6.8) 16 19 13 68.4 (43.4, 87.4) 94 63 67.0(56.6, 76.4) 52 0 0.0 (0.0, 6.8) 32 19 11 57.9 (33.5, 79.7) 94 53 56.4(45.8, 66.6) 52 0 0.0 (0.0, 6.8) 64 19 6 31.6 (12.6, 56.6) 94 23 24.5(16.2, 34.4) 52 0 0.0 (0.0, 6.8) 128 19 2 10.5  (1.3, 33.1) 94 10 10.6 (5.2, 18.7) 52 0 0.0 (0.0, 6.8) 12 to <18 Months 4 9 7 77.8 (40.0,97.2) 47 34 72.3 (57.4, 84.4) 23 0 0.0  (0.0, 14.8) 8 9 7 77.8 (40.0,97.2) 47 34 72.3 (57.4, 84.4) 23 0 0.0  (0.0, 14.8) 16 9 7 77.8 (40.0,97.2) 47 34 72.3 (57.4, 84.4) 23 0 0.0  (0.0, 14.8) 32 9 5 55.6 (21.2,86.3) 47 29 61.7 (46.4, 75.5) 23 0 0.0  (0.0, 14.8) 64 9 3 33.3  (7.5,70.1) 47 13 27.7 (15.6, 42.6) 23 0 0.0  (0.0, 14.8) 128 9 1 11.1  (0.3,48.2) 47 5 10.6  (3.5, 23.1) 23 0 0.0  (0.0, 14.8) 18 to <24 Months 4 107 70.0 (34.8, 93.3) 47 30 63.8 (48.5, 77.3) 29 0 0.0  (0.0, 11.9) 8 10 660.0 (26.2, 87.8) 47 30 63.8 (48.5, 77.3) 29 0 0.0  (0.0, 11.9) 16 10 660.0 (26.2, 87.8) 47 29 61.7 (46.4, 75.5) 29 0 0.0  (0.0, 11.9) 32 10 660.0 (26.2, 87.8) 47 24 51.1 (36.1, 65.9) 29 0 0.0  (0.0, 11.9) 64 10 330.0  (6.7, 65.2) 47 10 21.3 (10.7, 35.7) 29 0 0.0  (0.0, 11.9) 128 10 110.0  (0.3, 44.5) 47 5 10.6  (3.5, 23.1) 29 0 0.0  (0.0, 11.9) 1 Monthafter Vaccination 3 12 to <24 Months 4 19 17 89.5 (66.9, 98.7) 94 8287.2 (78.8, 93.2) 54 0 0.0 (0.0, 6.6) 8 19 17 89.5 (66.9, 98.7) 94 8186.2 (77.5, 92.4) 54 0 0.0 (0.0, 6.6) 16 19 16 84.2 (60.4, 96.6) 94 8186.2 (77.5, 92.4) 54 0 0.0 (0.0, 6.6) 32 19 12 63.2 (38.4, 83.7) 94 7276.6 (66.7, 84.7) 54 0 0.0 (0.0, 6.6) 64 19 7 36.8 (16.3, 61.6) 94 5558.5 (47.9, 68.6) 54 0 0.0 (0.0, 6.6) 128 19 4 21.1  (6.1, 45.6) 94 3031.9 (22.7, 42.3) 54 0 0.0 (0.0, 6.6) 12 to <18 Months 4 9 8 88.9 (51.8,99.7) 47 41 87.2 (74.3, 95.2) 24 0 0.0  (0.0, 14.2) 8 9 8 88.9 (51.8,99.7) 47 41 87.2 (74.3, 95.2) 24 0 0.0  (0.0, 14.2) 16 9 7 77.8 (40.0,97.2) 47 41 87.2 (74.3, 95.2) 24 0 0.0  (0.0, 14.2) 32 9 5 55.6 (21.2,86.3) 47 35 74.5 (59.7, 86.1) 24 0 0.0  (0.0, 14.2) 64 9 4 44.4 (13.7,78.8) 47 27 57.4 (42.2, 71.7) 24 0 0.0  (0.0, 14.2) 128 9 2 22.2  (2.8,60.0) 47 14 29.8 (17.3, 44.9) 24 0 0.0  (0.0, 14.2) 18 to <24 Months 410 9 90.0 (55.5, 99.7) 47 41 87.2 (74.3, 95.2) 30 0 0.0  (0.0, 11.6) 810 9 90.0 (55.5, 99.7) 47 40 85.1 (71.7, 93.8) 30 0 0.0  (0.0, 11.6) 1610 9 90.0 (55.5, 99.7) 47 40 85.1 (71.7, 93.8) 30 0 0.0  (0.0, 11.6) 3210 7 70.0 (34.8, 93.3) 47 37 78.7 (64.3, 89.3) 30 0 0.0  (0.0, 11.6) 6410 3 30.0  (6.7, 65.2) 47 28 59.6 (44.3, 73.6) 30 0 0.0  (0.0, 11.6) 12810 2 20.0  (2.5, 55.6) 47 16 34.0 (20.9, 49.3) 30 0 0.0  (0.0, 11.6)Abbreviations: hSBA = serum bactericidal assay using uman complement.^(a)N = number of subjects with valid and determinate hSBA titers forthe given strain. ^(b)n = Number of subjects with observed hSBA titer ≥the defined titer for the given strain at the given time point.^(c)Exact 2-sided CI based upon observed proportion of subjects, usingthe Clopper and Pearson method.

Exploratory Immunogenicity Endpoints

hSBA Titer ≥4-Fold Increase from Baseline Table 28 presents theproportion of subjects with hSBA titers with a ≥4-fold rise frombaseline for the 4 primary MnB test strains.

At 1 month after Vaccination 2, the proportion of subjects 12 to <18months of age achieving a ≥4-fold rise in hSBA titer from baseline was80.0% and 62.2% for PMB80 (A22); 100.0% and 97.9% for PMB2001 (A56);60.0% and 19.0% for PMB2948 (B24); and 77.8% and 70.2% for PMB2707 (B44)for the 60-μg and 120-μg groups, respectively. For the 18 to <24 monthsage stratum, the proportion of subjects achieving a ≥4-fold rise in hSBAtiter from baseline was 66.7% and 80.0% for PMB80 (A22); 90.0% and100.0% for PMB2001 (A56); 33.3% and 40.9% for PMB2948 (B24); and 60.0%and 61.7% for PMB2707 (B44) for the 60-μg and 120-μg groups,respectively. Overall, for the combined age stratum, the proportion ofsubjects achieving a ≥4-fold rise in hSBA titer from baseline at 1 monthafter Vaccination 2 was 73.7% and 71.6% for PMB80 (A22); 94.7% and 98.9%for PMB2001 (A56); 47.4% and 30.2% for PMB2948 (B24); and 68.4% and66.0% for PMB2707 (B44) for the 60-μg and 120-μg groups, respectively.

At 1 month after Vaccination 3, the proportion of subjects 12 to <18months of age achieving a ≥4-fold rise in hSBA titer from baseline was77.8% and 86.7% for PMB80 (A22); 100.0% and 95.7% for PMB2001 (A56);88.9% and 66.7% for PMB2948 (B24); and 77.8% and 85.1% for PMB2707 (B44)for the 60-μg and 120-μg groups, respectively. For the 18 to <24 monthsage stratum, the proportion of subjects achieving a ≥4-fold rise in hSBAtiter from baseline was 90.9% and 88.2% for PMB80 (A22); 100.0% and100.0% for PMB2001 (A56); 63.6% and 68.0% for PMB2948 (B24); and 90.0%and 85.1% for PMB2707 (B44) for the 60-μg and 120-μg groups,respectively. Overall, for the combined age stratum, the proportion ofsubjects achieving a ≥4-fold rise in hSBA titer from baseline at 1 monthafter Vaccination 3 was 85.0% and 87.5% for PMB80 (A22); 100.0% and97.9% for PMB2001 (A56); 75.0% and 67.4% for PMB2948 (B24); and 84.2%and 85.1% for PMB2707 (B44) for the 60-μg and 120-μg groups,respectively.

The proportion of subjects in the HAV/saline group (combined agestratum) achieving a ≥4-fold rise in hSBA titer at 1 month afterVaccination 3 was 5.0% for PMB80 (A22), 1.9% for PMB2001 (A56), 3.3% forPMB2948 (B24), and 0.0% for PMB2707 (B44).

TABLE 28 Subjects Achieving ≥4-Fold Rise in hSBA Titer for PrimaryStrains—Evaluable Immunogenicity Population Strain (Variant) VaccineGroup (as Randomized) Sampling Time Point 60 μg rLP2086 120 μg rLP2086HAV/Saline Age Strata N^(a) n^(b) (%) (%) (95% CI)^(c) N^(a) n^(b) (%)(%) (95% CI)^(c) N^(a) n^(b) (%) (%) (95% CI)^(c) PMB80 (A22) 1 Monthafter Vaccination 2 12 to <24 Months 19 14 (73.7) (48.8, 90.9) 95 68(71.6) (61.4, 80.4) 59 1 (1.7) (0.0, 9.1) 12 to <18 Months 10 8 (80.0)(44.4, 97.5) 45 28 (62.2) (46.5, 76.2) 30 0 (0.0)  (0.0, 11.6) 18 to <24Months 9 6 (66.7) (29.9, 92.5) 50 40 (80.0) (66.3, 90.0) 29 1 (3.4) (0.1, 17.8) 1 Month after Vaccination 3 12 to <24 Months 20 17 (85.0)(62.1, 96.8) 96 84 (87.5) (79.2, 93.4) 60 3 (5.0) (1.0, 13.9) 12 to <18Months 9 7 (77.8) (40.0, 97.2) 45 39 (86.7) (73.2, 94.9) 31 1 (3.2) (0.1, 16.7) 18 to <24 Months 11 10 (90.9) (58.7, 99.8) 51 45 (88.2)(76.1, 95.6) 29 2 (6.9)  (0.8, 22.8) PMB2001 (A56) 1 Month afterVaccination 2 12 to <24 Months 19 18 (94.7) (74.0, 99.9) 95 94 (8.9) (94.3, 100.0) 52 0 (0.0) (0.0, 6.8) 12 to <18 Months 9 9 (100.0)  (66.4, 100.0) 47 46 (97.9) (88.7, 99.9) 23 0 (0.0)  (0.0, 14.8) 18 to<24 Months 10 9 (90.0) (55.5, 99.7) 48 48 (100.0)   (92.6, 100.0) 29 0(0.0)  (0.0, 11.9) 1 Month after Vaccination 3 12 to <24 Months 19 19(100.0)   (82.4, 100.0) 95 93 (97.9) (92.6, 99.7) 54 1 (1.9) (0.0, 9.9)12 to <18 Months 9 9 (100.0)   (66.4, 100.0) 47 45 (95.7) (85.5, 99.5)24 0 (0.0)  (0.0, 14.2) 18 to <24 Months 10 10 (100.0)   (69.2, 100.0)48 48 (100.0)   (92.6, 100.0) 30 1 (3.3)  (0.1, 17.2) PMB2948 (B24) 1Month after Vaccination 2 12 to <24 Months 19 9 (47.4) (24.4, 71.1) 8626 (30.2) (20.8, 41.1) 59 0 (0.0) (0.0, 6.1) 12 to <18 Months 10 6(60.0) (26.2, 87.8) 42 8 (19.0)  (8.6, 34.1) 30 0 (0.0)  (0.0, 11.6) 18to <24 Months 9 3 (33.3)  (7.5, 70.1) 44 18 (40.9) (26.3, 56.8) 29 0(0.0)  (0.0, 11.9) 1 Month after Vaccination 3 12 to <24 Months 20 15(75.0) (50.9, 91.3) 95 64 (67.4) (57.0, 76.6) 60 2 (3.3)  (0.4, 11.5) 12to <18 Months 9 8 (88.9) (51.8, 99.7) 45 30 (66.7) (51.0, 80.0) 31 1(3.2)  (0.1, 16.7) 18 to <24 Months 11 7 (63.6) (30.8, 89.1) 50 34(68.0) (53.3, 80.5) 29 1 (3.4)  (0.1, 17.8)

TABLE 28 Subjects Achieving ≥4-Fold Rise in hSBA Titer for PrimaryStrains—Evaluable Immunogenicity Population Strain (Variant) VaccineGroup (as Randomized) Sampling Time Point 60 μg rLP2086 120 μg rLP2086HAV/Saline Age Strata N^(a) n^(b) (%) (%) (95% CI)^(c) N^(a) n^(b) (%)(%) (95% CI)^(c) N^(a) n^(b) (%) (%) (95% CI)^(c) PMB2707 (B44) 1 Monthafter Vaccination 2 12 to <24 Months 19 13 (68.4) (43.4, 87.4) 94 62(66.0) (55.5, 75.4) 52 0 (0.0) (0.0, 6.8) 12 to <18 Months 9 7 (77.8)(40.0, 97.2) 47 33 (70.2) (55.1, 82.7) 23 0 (0.0)  (0.0, 14.8) 18 to <24Months 10 6 (60.0) (26.2, 87.8) 47 29 (61.7) (46.4, 75.5) 29 0 (0.0) (0.0, 11.9) 1 Month after Vaccination 3 12 to <24 Months 19 16 (84.2)(60.4, 96.6) 94 80 (85.1) (76.3, 91.6) 54 0 (0.0) (0.0, 6.6) 12 to <18Months 9 7 (77.8) (40.0, 97.2) 47 40 (85.1) (71.7, 93.8) 24 0 (0.0) (0.0, 14.2) 18 to <24 Months 10 9 (90.0) (55.5, 99.7) 47 40 (85.1)(71.7, 93.8) 30 0 (0.0)  (0.0, 11.6) Abbreviations: hSBA = serumbactericidal assay using human complement; LLOQ = lower limit ofquantitation; LOD = limit of detection; Note: LLOQ = 1:16 for A22; 1:8for A56, B24, and B44. Note: The 4-fold increase is defined as follows:(1) For subjects with a baseline hSBA titer below the LOD (hSBA titer<1:4), a response is defined as an hSBA titer ≥1:16 or the LLOQ(whichever titer is higher). (2) For subjects with a baseline hSBA titer≥LOD and <LLOQ, a response is defined as an hSBA titer ≥4 times theLLOQ. (3) For subjects with a baseline hSBA titer ≥LLOQ, a response isdefined as an hSBA titer ≥4 times the baseline titer. ^(a)For hSBA titerfold rise ≥4 from baseline, N = number of subjects with valid anddeterminate hSBA titers for the given strain at both the specified timepoint and baseline. ^(b)For hSBA titer fold rise ≥4 from baseline, n =number of subjects who achieved hSBA titer fold rise ≥4 from baselinefor the given strain. ^(c)Exact 2-sided CI based upon observedproportion of subjects, using the Clopper and Pearson method.

Similar results were observed for the mITT population.

Subgroup analyses of the proportion of subjects achieving a ≥4-fold risein hSBA titer for each of the 4 primary MnB test strains were assessedfor the evaluable immunogenicity population by sex and country. Therewere no clinically important differences observed in the subgroupanalyses performed.

Reverse Cumulative Distribution Curves for the Primary MnB Test Strains

The RCDCs of the proportions of subjects exhibiting an hSBA response(≥LLOQ) for each of the 4 primary MnB test strains and at each samplingtime point, for the combined age stratum were assessed for PMB80 [A22],PMB2001 [A56], PMB2948 [B24], and PMB2707 [B44]. The RCDCs showed thatthe immune responses were higher after Vaccination 2 and Vaccination 3for the bivalent rLP2086 groups versus the HAV/saline group. The immuneresponses for the bivalent rLP2086 groups increased with eachvaccination.

Immunogenicity Conclusions

The primary objectives of this study were to describe the immuneresponse to bivalent rLP2086 as measured by hSBA against 4 primary MnBtest strains, 2 expressing an LP2086 subfamily A protein and 2expressing an LP2086 subfamily B protein, measured 1 month after thethird vaccination in healthy subjects 12 to <18 months of age and 18 to<24 months of age. The description of immune responses for the combinedage stratum (12 to <24 months) was a secondary objective. The primaryendpoints for the primary objectives were the proportions of subjects ineach age stratum achieving hSBA titers ≥LLOQ for each of the 4 primaryMnB strains 1 month after the third vaccination.

A robust immune response was observed at both dose levels for toddlers12 to <18 months of age and for toddlers 18 to <24 months of age, aswell as for the combined age stratum (12 to <24 months) 1 month afterthe third dose of bivalent rLP2086, as confirmed by the proportion ofsubjects achieving an hSBA titer ≥LLOQ (1:8 for A56, B24 and B44; 1:16for A22) for each of the 4 primary MnB test strains. For the 60-μggroup, the proportion of subjects achieving an hSBA titer ≥LLOQ rangedfrom 88.9% to 100.0% for the younger toddlers (12 to <18 months) andfrom 81.8% to 100.0% for the older toddlers (18 to <24 months) after 3doses. For the 120-μg group, the proportion of subjects achieving anhSBA titer ≥LLOQ ranged from 71.1% to 100.0% for toddlers 12 to <18months of age and from 72.0% to 100.0% for toddlers 18 to <24 months ofage after 3 doses. For the combined age stratum the proportion ofsubjects achieving an hSBA titer ≥LLOQ for each of the 4 primary MnBtest strains 1 month after the third vaccination ranged from 85.0% to100.0% for the 60-μg group and from 71.6% to 100.0% for the 120-μggroup. These findings are further supported by increases in GMTs (rangefrom 4.0 to 8.5 at baseline to 15.1 to 171.4 at 1 month afterVaccination 3) and in the proportion of subjects achieving an hSBA titer≥1:4 (71.1% to 100.0%) or ≥1:16 (63.6% to 100.0%) against each of the 4primary MnB test strains after 3 doses of bivalent rLP2086 compared tobaseline across both age strata and dose levels. Additionally, theproportion of subjects for the combined age stratum achieving an hSBAfold rise 4 from baseline to 1 month after the third vaccination foreach of the 4 primary MnB test strains ranged from 67.4% to 100.0% forboth dose levels. In conclusion, 3 doses of either 60 μg or 120 μg ofbivalent rLP2086 administered on a 0-, 2-, 6-month schedule, inducedrobust immune responses in toddlers 12 to <24 months of age (bothindividual and combined age strata).

The secondary objective of the study was to describe immune responses 1month after the second dose of bivalent rLP2086, as assessed by ≥LLOQresponses, defined hSBA titers, and hSBA GMTs for the 2 age strata andthe combined age stratum. For the combined age stratum, the proportionof subjects achieving an hSBA titer ≥LLOQ after the second dose ofbivalent rLP2086 (administered 2 months after the first dose) rangedfrom 57.9% to 94.7% for subjects in the 60-μg group and from 33.7% to100.0% for subjects in the 120-μg group. Similar results were obtainedfor the 2 individual age strata with no clinically meaningfuldifferences between the younger and older age strata. These findings aresupported by increases in GMTs (range 7.2 to 110.6) over baseline and inthe proportion of subjects achieving an hSBA titer ≥1:4 (36.0% to100.0%) or ≥1:16 (32.6% to 100%) against each of the 4 primary MnB teststrains after 2 doses of bivalent rLP2086 compared to baseline acrossboth dose levels for the combined age stratum. Similar results wereobtained for the 2 individual age strata.

Additionally, the proportion of subjects for the combined age stratumachieving an hSBA fold rise ≥4 from baseline to 1 month after the secondvaccination for each of the 4 primary MnB test strains ranged from 30.2%to 98.9%. In conclusion, 2 doses of either 60 μg or 120 μg of bivalentrLP2086 administered 2 months apart induced immune responses in toddlers12 to <24 months of age (both individual and combined age strata).

In summary, at both the 60-μg and 120-μg dose levels, bivalent rLP2086given as 3 doses on a 0-, 2-, and 6-month schedule elicits a robustimmune response among toddlers 12 to <24 months of age with protectiveantibody titers achieved as measured by hSBA in a high proportion ofsubjects after the third dose.

Discussion and Overall Conclusions

Immunogenicity Discussion. Immunogenicity results from this Phase 2study of a 3-dose regimen (0-, 2-, and 6-month schedule) of bivalentrLP2086 (at 2 dose levels) given to toddlers 12 to <24 months of age areconsistent with previous studies in adolescents and young adults at the120-μg dose level.

Immunogenicity responses to bivalent rLP2086 vaccination were measuredin validated hSBAs using 4 primary MnB test strains, each expressingfHBP variants heterologous to the vaccine component antigens, usingcriteria more stringent than the accepted correlate of protection (hSBAtiter ≥1:4). Based on an hSBA titer ≥LLOQ for the 4 primary MnB teststrains 1 month after Vaccination 3, the toddlers participating in thisstudy (at either dose level) had similar immune responses compared toadolescents (10 years to <19 years) participating in Study B1971009 andtoddlers and children participating in Study B1971017 (≥24 months to <10years), with proportions of subjects achieving an hSBA titer ≥LLOQ afterthe third vaccination (0-, 2-, 6-month schedule) ranging from 71.6% to100.0% for the 120-μg group (12 to <24 months age) in this study, 87.1%to 99.5% in Study B1971009, and 79.1% to 100.0% in Study B1971017.Clinically meaningful differences in the proportion of subjectsachieving an hSBA titer ≥LLOQ for the 4 primary MnB test strains 1 monthafter Vaccination 3 between these 3 studies are not apparent, despitethe fact that Study B1971009 had a much higher proportion of adolescentsubjects with a prevaccination hSBA titer ≥LLOQ compared to the toddlersin this study. Bivalent rLP2086 appears to be highly immunogenic in the12 to <24 months age population and is likely to offer protectionagainst MnB infection similarly to that expected for adolescents basedon the hSBA correlate of protection. After 3 doses for the individualage strata and for both age strata combined, responses to the 60-μg doselevel were not meaningfully different than responses to the 120-μg doselevel.

With regard to the secondary objectives, immune responses in this studyfor the combined age stratum (12 to <24 months) 1 month after the seconddose of bivalent rLP2086 (either dose level), the proportion of subjectsachieving an hSBA titer ≥LLOQ ranged from 33.7% to 100.0% compared toadolescents (10 years to <19 years) participating in Study B1971009receiving 2 doses of bivalent rLP2086 given 2 months apart, which rangedfrom 64.2% to 99.1% and toddlers and children (≥24 months to <10 years)participating in Study B1971017 which ranged from 48.5% to 100.0%. Forboth age strata and the combined age stratum, immune responses after 2doses of 60 μg bivalent rLP2086 were not meaningfully different thanresponses following 2 doses of 120 μg bivalent rLP2086. It should benoted that the smaller sample size in the 60-μg group make definitiveconclusions difficult when comparing response rates between 60-μg and120-μg dose levels.

In summary, at both the 60-μg and 120-μg dose levels, bivalent rLP2086administered on a 0-, 2-, and 6-month schedule is highly immunogenicamong toddlers 12 to <24 months of age with protective immune responsesachieved as measured by hSBA in a high proportion of subjects after thethird dose. Immune responses, as measured in this study, appear to besimilar to that observed in prior studies among adolescents and children1 month after the third dose. The 3-dose regimen appears to provide highrates of protective immunity in toddlers 12 to <24 months of age.

Overall Conclusions. In conclusion, the 60-μg and 120-μg dose levels ofbivalent rLP2086 when administered to toddlers 12 to <24 months of ageon a 0-, 2-, and 6-month schedule elicit protective antibody titersafter the third dose as measured by hSBAs. The vaccine, as administeredin this study, was safe and well tolerated with an acceptable safetyprofile for toddlers 12 to <24 months of age.

Example 22: Assessment of the Neisseria meningitidis Serogroup BImmunogenicity of Mn Pentavalent and Trumenba® Vaccines in CBA/J Mice

The immune response to Neisseria meningitidis serogroup B fHBP followingvaccination with either bivalent Mn B fHBP vaccine, Trumenba, or thebivalent Mn B fHBP vaccine formulated with quadrivalent ACWYpolysaccharide conjugate vaccine (Mn Pentavalent ABCWY) was evaluated inCBA/J mice. Groups of CBA/J mice were immunized with 3 differentvaccines: Pentavalent (ABCYW), Trumenba® (MnB) and Nimenrix (ACYW)(Table 29).

TABLE 29 Study Design: Dose Levels for each Vaccine Dose Levels, μg/0.25mL Dose Dilution Mn Pentavalent TRUMENBA ® NIMENRIX ® AlPO₄ Factor(ABCWY) (B) (ACWY) (diluent) 1 8 + 1.33 8 1.33 125 2 4 + 0.67 4 0.67 1254 2 + 0.33 2 0.33 125 8 1 + 0.17 1 0.17 125

For each arm, CBA/J mice (25/group) were subcutaneously immunized in thescruff of the neck using 2-fold dilution dose levels of the respectivevaccine (Table 29). Mice were primed with the vaccine at time 0 andboosted at week 2. Sera were collected PD2 at week 3 for testing usingtwo different serum bactericidal assays that utilized human complement(hSBA). One hSBA used an fHBP subfamily A expressing strain (M98250771)and the other an fHBP subfamily B expressing strain (CDC1127).

The hSBA measures antibody-dependent, complement mediated bactericidalactivity against N meningitidis serogroup B strains. Briefly, test seraat the appropriate dilution were mixed in 96-well microtiter assayplates with freshly prepared bacterial cultures of the N meningitidis Bstrains (subfamily A or B) and human complement. Assay plates wereplaced on an orbital shaker and mixed for 30 min in a humidifiedincubator (37° C./5% CO₂). Subsequently, aliquots of the assay reactionfrom each well were transferred to 96-well filter plates for enumerationof surviving bacteria.

Response rates to vaccination were calculated as the percentage of micein each dosing group (n=25) that respond in hSBAs. When tested at apredetermined dilution level, mouse serum samples that kill ≥50% of theT₃₀ control meningococcal bacteria are considered responders. The T₃₀control wells contain bacteria and complement but no test serum and arecounted at the end of the 30 minute assay incubation.

Table 30 and Table 31 show comparable dose-dependent response ratesinduced by either TRUMENBA or Mn Pentavalent for both subfamily A andsubfamily B of the N meningitidis serotype B strains. As expected,NIMENRIX™ did not induce a functional immune response to Mn B strains.

TABLE 30 Subfamily A hSBA responses (% responders) Dilution Factor ^(a)TRUMENBA NIMENRIX Penta 8 24% 0%  8% 4 40% 0% 16% 2 52% 0% 56% 1 80% 0%92% ^(a) See corresponding dose levels in Table 29

TABLE 31 Subfamily B hSBA responses % responders) Dilution Factor ^(a)TRUMENBA NIMENRIX Penta 8 28% 0% 36% 4 56% 0% 60% 2 60% 0% 76% 1 72% 0%76% ^(a) See corresponding dose levels in Table 29

The following clauses describe additional embodiments of the invention:

-   -   C1. A composition comprising a polypeptide and a Neisseria        meningitidis serogroup A (MenA) capsular saccharide conjugate; a        Neisseria meningitidis serogroup C (MenC) capsular saccharide        conjugate; a Neisseria meningitidis serogroup W₁₃₅ (MenW)        capsular saccharide conjugate; and a Neisseria meningitidis        serogroup Y (MenY) capsular saccharide conjugate.    -   C2. The composition of clause C1, wherein the MenA capsular        saccharide is conjugated to a carrier protein; the MenC capsular        saccharide is conjugated to a carrier protein; the MenW capsular        saccharide is conjugated to a carrier protein; and the MenY        capsular saccharide is conjugated to a carrier protein.    -   C3. The composition of clause C1, wherein the composition        further includes a Neisseria meningitidis serogroup X (MenX)        capsular saccharide conjugate.    -   C4. The composition of clause C1, wherein the polypeptide is a        factor H binding protein (fHBP).    -   C5. The composition of clause C1, wherein the polypeptide        comprises an amino acid sequence having at least 70% identity to        the amino acid sequence set forth in any one of SEQ ID NO: 1,        SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID        NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:        13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17,        SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ        ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID        NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO:        32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36,        SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ        ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID        NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO:        49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53,        SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ        ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, and SEQ        ID NO: 62.    -   C6. A composition comprising        -   a. a first lipidated polypeptide comprising the amino acid            sequence set forth in SEQ ID NO: 1;        -   b. a second lipidated polypeptide comprising the amino acid            sequence set forth in SEQ ID NO: 2; and        -   c. a Neisseria meningitidis serogroup A (MenA) capsular            saccharide conjugated to a carrier protein.    -   C7. A composition comprising        -   a. a first lipidated polypeptide comprising the amino acid            sequence set forth in SEQ ID NO: 1;        -   b. a second lipidated polypeptide comprising the amino acid            sequence set forth in SEQ ID NO: 2; and        -   c. a Neisseria meningitidis serogroup C (MenC) capsular            saccharide conjugated to a carrier protein.    -   C8. A composition comprising        -   a. a first lipidated polypeptide comprising the amino acid            sequence set forth in SEQ ID NO:1;        -   b. a second lipidated polypeptide comprising the amino acid            sequence set forth in SEQ ID NO: 2; and        -   c. a Neisseria meningitidis serogroup W-135 (MenW) capsular            saccharide conjugated to a carrier protein.    -   C9. A composition comprising        -   a. a first lipidated polypeptide comprising the amino acid            sequence set forth in SEQ ID NO: 1;        -   b. a second lipidated polypeptide comprising the amino acid            sequence set forth in SEQ ID NO: 2; and        -   c. a Neisseria meningitidis serogroup Y (MenY) capsular            saccharide conjugated to a carrier protein.    -   C10. A composition comprising        -   a. a first lipidated polypeptide comprising the amino acid            sequence set forth in SEQ ID NO: 1;        -   b. a second lipidated polypeptide comprising the amino acid            sequence set forth in SEQ ID NO: 2;        -   c. a Neisseria meningitidis serogroup A (MenA) capsular            saccharide conjugated to a carrier protein;        -   d. a Neisseria meningitidis serogroup C (MenC) capsular            saccharide conjugated to a carrier protein;        -   e. a Neisseria meningitidis serogroup W₁₃₅ (MenW) capsular            saccharide conjugated to a carrier protein; and        -   f. a Neisseria meningitidis serogroup Y (MenY) capsular            saccharide conjugated to a carrier protein.    -   C11. A composition comprising        -   a. a first lipidated polypeptide comprising the amino acid            sequence set forth in SEQ ID NO: 1;        -   b. a second lipidated polypeptide comprising the amino acid            sequence set forth in SEQ ID NO: 2;        -   c. a Neisseria meningitidis serogroup A (MenA) capsular            saccharide conjugated to tetanus toxoid carrier protein            (TT);        -   d. a Neisseria meningitidis serogroup C (MenC) capsular            saccharide conjugated to tetanus toxoid carrier protein            (TT);        -   e. a Neisseria meningitidis serogroup W₁₃₅ (MenW) capsular            saccharide conjugated to tetanus toxoid carrier protein            (TT); and        -   f. a Neisseria meningitidis serogroup Y (MenY) capsular            saccharide conjugated to tetanus toxoid carrier protein            (TT).    -   C12. The composition according to clause C1, wherein the MenA        capsular saccharide is conjugated to an adipic acid dihydrazide        (ADH) linker by 1-cyano-4-dimethylamino pyridinium        tetrafluoroborate chemistry, and wherein the linker is        conjugated to tetanus toxoid carrier protein (TT) by        carbodiimide chemistry (MenA_(AH)-TT conjugate).    -   C13. The composition according to clause C1, wherein the MenC        capsular saccharide is conjugated to an ADH linker by        1-cyano-4-dimethylamino pyridinium tetrafluoroborate chemistry,        and wherein the linker is conjugated to tetanus toxoid carrier        protein (TT) by carbodiimide chemistry (MenC_(AH)-TT conjugate).    -   C14. The composition according to clause C1, wherein the MenW        capsular saccharide is directly conjugated to tetanus toxoid        carrier protein (TT) by 1-cyano-4-dimethylamino pyridinium        tetrafluoroborate chemistry, in the absence of a linker (MenW-TT        conjugate).    -   C15. The composition according to clause C1, wherein the MenY        capsular saccharide is directly conjugated to tetanus toxoid        carrier protein (TT) by 1-cyano-4-dimethylamino pyridinium        tetrafluoroborate chemistry, in the absence of a linker (MenY-TT        conjugate).    -   C16. The composition according to clause C1, further comprising        Tris-HCl.    -   C17. The composition according to clause C1, further comprising        sodium chloride.    -   C18. The composition according to clause C1, further comprising        sucrose.    -   C19. The composition according to clause C1, further comprising        Histidine.    -   C20. The composition according to clause C1, further comprising        polysorbate 80.    -   C21. The composition according to clause C1, further comprising        aluminum.    -   C22. The composition according to clause C1, further comprising        aluminum phosphate.    -   C23. The composition according to clause C1, wherein the        composition comprises about 120 μg/ml of the first polypeptide;        about 120 μg/ml of the second polypeptide; about 0.5 mg/ml        aluminum as aluminum phosphate; about 0.02 mg polysorbate-80;        about 10 mM histidine; and about 150 mM sodium chloride.    -   C24. The composition according to clause C1, wherein the        composition comprises about 60 μg of the first polypeptide;        about 60 μg of the second polypeptide; about 5 μg of the MenA        capsular saccharide conjugated to about 7.5 μg TT; about 5 μg of        the MenC capsular saccharide conjugated to about 7.5 μg TT;        about 5 μg of the MenW capsular saccharide conjugated to about        3.75 μg TT; about 5 μg of the MenY capsular saccharide        conjugated to about 3.25 μg TT; about 97 μg Tris-HCl, pH        6.8±0.3; 4.69-4.71 mg of sodium chloride; about 28 mg of        sucrose; about 0.78 mg of L-Histidine; about 0.02 mg        polysorbate-80; about 0.25 mg aluminum; and further comprising        0.5 mL water, per dose.    -   C25. The composition according to clause C1, wherein the        composition is suitable for use in a patient aged 12 to <18        Months or 18 to <24 Months.    -   C26. The composition according to clause C1, wherein the        composition is suitable for use in a patient aged 18 to <24        Months.    -   C27. The composition according to clause C1, wherein the        composition is suitable for use in a patient aged ≥24 Months to        <10 Years.    -   C28. The composition according to clause C20, wherein the        composition comprises at least 0.010 mg polysorbate-80 and at        most 0.018 mg polysorbate-80.    -   C29. The composition according to clause C20, wherein the        composition comprises at least 0.01 mg polysorbate-80 and at        most 0.02 mg polysorbate-80.    -   C30. The composition according to clause C1, wherein the        composition does not further comprise a polypeptide having less        than 100% sequence identity to SEQ ID NO: 1.    -   C31. The composition according to clause C1, wherein the first        polypeptide has a total of 258 amino acids.    -   C32. The composition according to clause C1, wherein the        composition does not further comprise a polypeptide having less        than 100% sequence identity to SEQ ID NO: 2.    -   C33. The composition according to clause C1, wherein the second        polypeptide has a total of 261 amino acids.    -   C34. The composition according to clause C1, wherein the        composition comprises at most two lipidated polypeptides.    -   C35. The composition according to clause C1, wherein the        composition does not comprise a hybrid protein.    -   C36. The composition according to clause C1, wherein the        composition does not comprise a chimeric protein.    -   C37. The composition according to clause C1, wherein the        composition does not comprise a fusion protein.    -   C38. The composition according to clause C1, wherein the        composition is not lyophilized.    -   C39. The composition according to clause C1, wherein the        composition does not comprise formaldehyde.    -   C40. The composition according to clause C1, wherein the        composition does not comprise diphtheria toxoid or CRM.    -   C41. The composition according to clause C1, wherein the        composition does not comprise a MenA capsular saccharide in the        absence of an adipic acid dihydrazide (ADH) linker.    -   C42. The composition according to clause C1, wherein the        composition does not comprise a MenC capsular saccharide in the        absence of an adipic acid dihydrazide (ADH) linker.    -   C43. The composition according to clause C1, wherein the        composition is a liquid composition.    -   C44. The composition according to clause C1, wherein the        composition does not further comprise any one of the following        immunogenic compositions: MENACTRA®, MENVEO®, ADACEL®, HAVRIX®,        GARDASIL®, REPEVAX, or any combination thereof.    -   C45. The composition according to clause C1, wherein the        composition does not further comprise a meningococcal A, C, Y        and W-135 polysaccharide conjugate (MCV4) composition, wherein        the carrier protein is diphtheria toxoid.    -   C46. The composition according to clause C1, wherein the        composition does not further comprise a meningococcal A, C, Y        and W-135 polysaccharide conjugate (MCV4) composition, wherein        the carrier protein is CRM₁₉₇.    -   C47. The composition according to clause C1, wherein the        composition does not further comprise a NIMENRIX vaccine.    -   C48. The composition according to clause C1, wherein the        composition does not further comprise a NIMENRIX vaccine,        wherein NIMENRIX comprises of a diluent consisting of sodium        chloride and water.    -   C49. A kit comprising (a) a first composition comprising a        lipidated MenB rLP2086 subfamily A polypeptide and a lipidated        MenB rLP2086 subfamily B polypeptide; and (b) a second        composition comprising a Neisseria meningitidis serogroup A        (MenA) capsular saccharide conjugated to tetanus toxoid carrier        protein (TT); a Neisseria meningitidis serogroup C (MenC)        capsular saccharide conjugated to tetanus toxoid carrier protein        (TT); a Neisseria meningitidis serogroup W₁₃₅ (MenW) capsular        saccharide conjugated to tetanus toxoid carrier protein (TT);        and a Neisseria meningitidis serogroup Y (MenY) capsular        saccharide conjugated to tetanus toxoid carrier protein (TT).    -   C50. The kit according to clause C49, wherein the first        composition is a liquid composition and the second composition        is a lyophilized composition.    -   C51. The kit according to clause C49, wherein the lyophilized        composition does not comprise polysorbate 80.    -   C52. The kit according to clause C49, wherein the kit does not        further comprise any one of the following immunogenic        compositions: MENACTRA®, MENVEO®, ADACEL®, HAVRIX®, GARDASIL®,        REPEVAX, or any combination thereof.    -   C53. The kit according to clause C49, wherein the kit does not        further comprise a meningococcal A, C, Y and W-135        polysaccharide conjugate (MCV4) composition, wherein the carrier        protein is diphtheria toxoid.    -   C54. The kit according to clause C49, wherein the kit does not        further comprise a meningococcal A, C, Y and W-135        polysaccharide conjugate (MCV4) composition, wherein the carrier        protein is CRM₁₉₇.    -   C55. The kit according to clause C49, wherein the kit does not        further comprise a NIMENRIX vaccine.    -   C56. The kit according to clause C49, wherein the kit does not        further comprise a NIMENRIX® vaccine, wherein NIMENRIX®        comprises of a diluent consisting of sodium chloride and water.    -   C57. A kit comprising:        -   a. a liquid composition comprising            -   i. a first lipidated polypeptide comprising the amino                acid sequence set forth in SEQ ID NO: 1; and            -   ii. a second lipidated polypeptide comprising the amino                acid sequence set forth in SEQ ID NO: 2; and        -   b. a lyophilized composition comprising            -   i. a Neisseria meningitidis serogroup A (MenA) capsular                saccharide conjugated to an adipic acid dihydrazide                (ADH) linker by 1-cyano-4-dimethylamino pyridinium                tetrafluoroborate chemistry, wherein the linker is                conjugated to tetanus toxoid carrier protein (TT) by                carbodiimide chemistry (MenA_(AH)-TT conjugate);            -   ii. a Neisseria meningitidis serogroup C (MenC) capsular                saccharide conjugated to an ADH linker by                1-cyano-4-dimethylamino pyridinium tetrafluoroborate                chemistry, wherein the linker is conjugated to tetanus                toxoid carrier protein (TT) by carbodiimide chemistry                (MenC_(AH)-TT conjugate);            -   iii. a Neisseria meningitidis serogroup W₁₃₅ (MenW)                capsular saccharide directly conjugated to tetanus                toxoid carrier protein (TT) by 1-cyano-4-dimethylamino                pyridinium tetrafluoroborate chemistry, in the absence                of a linker (MenW-TT conjugate); and            -   iv. a Neisseria meningitidis serogroup Y (MenY) capsular                saccharide directly conjugated to tetanus toxoid carrier                protein (TT) by 1-cyano-4-dimethylamino pyridinium                tetrafluoroborate chemistry, in the absence of a linker                (MenY-TT conjugate).    -   C58. The kit according to clause C57, wherein the liquid        composition further comprises sodium chloride.    -   C59. The kit according to clause C57, wherein the liquid        composition further comprises L-Histidine.    -   C60. The kit according to clause C57, wherein the liquid        composition further comprises polysorbate 80.    -   C61. The kit according to clause C57, wherein the liquid        composition further comprises aluminum phosphate.    -   C62. The kit according to clause C57, wherein the liquid        composition does not further comprise Tris-HCl.    -   C63. The kit according to clause C57, wherein the liquid        composition does not further comprise sucrose.    -   C64. The kit according to clause C57, wherein the lyophilized        composition further comprises sodium chloride.    -   C65. The kit according to clause C57, wherein the lyophilized        composition does not comprise polysorbate-80.    -   C66. The kit according to clause C60, wherein the liquid        composition comprises at least 0.010 mg polysorbate-80 and at        most 0.018 mg polysorbate-80.    -   C67. The kit according to clause C60, wherein the liquid        composition comprises at least 0.010 mg polysorbate-80 and at        most 0.02 mg polysorbate-80.    -   C68. An immunogenic composition comprising:        -   a. a liquid composition comprising (i) a first lipidated            polypeptide comprising the amino acid sequence set forth in            SEQ ID NO: 1; and (ii) a second lipidated polypeptide            comprising the amino acid sequence set forth in SEQ ID NO:            2; and        -   b. a lyophilized composition comprising            -   i. a Neisseria meningitidis serogroup A (MenA) capsular                saccharide conjugated to an adipic acid dihydrazide                (ADH) linker by 1-cyano-4-dimethylamino pyridinium                tetrafluoroborate chemistry, wherein the linker is                conjugated to tetanus toxoid carrier protein (TT) by                carbodiimide chemistry (MenA_(AH)-TT conjugate);            -   ii. a Neisseria meningitidis serogroup C (MenC) capsular                saccharide conjugated to an ADH linker by                1-cyano-4-dimethylamino pyridinium tetrafluoroborate                chemistry, wherein the linker is conjugated to tetanus                toxoid carrier protein (TT) by carbodiimide chemistry                (MenC_(AH)-TT conjugate);            -   iii. a Neisseria meningitidis serogroup W₁₃₅ (MenW)                capsular saccharide directly conjugated to tetanus                toxoid carrier protein (TT) by 1-cyano-4-dimethylamino                pyridinium tetrafluoroborate chemistry, in the absence                of a linker (MenW-TT conjugate); and            -   iv. a Neisseria meningitidis serogroup Y (MenY) capsular                saccharide directly conjugated to tetanus toxoid carrier                protein (TT) by 1-cyano-4-dimethylamino pyridinium                tetrafluoroborate chemistry, in the absence of a linker                (MenY-TT conjugate).    -   C69. The immunogenic composition according to clause C68,        wherein the lyophilized composition is reconstituted with the        liquid composition.    -   C70. The immunogenic composition according to clause C68,        wherein the liquid composition further comprises histidine.    -   C71. The immunogenic composition according to clause C68,        wherein the liquid composition further comprises polysorbate-80.    -   C72. The immunogenic composition according to clause C68,        wherein the liquid composition further comprises aluminum        phosphate.    -   C73. The immunogenic composition according to clause C68,        wherein the liquid composition further comprises sodium        chloride.    -   C74. The immunogenic composition according to clause C68,        wherein the composition is suitable for use in a patient aged 12        to <18 Months or 18 to <24 Months.    -   C75. The immunogenic composition according to clause C68,        wherein the composition is suitable for use in a patient aged 18        to <24 Months.    -   C76. The immunogenic composition according to clause C68,        wherein the composition is suitable for use in a patient aged        ≥24 Months to <10 Years.    -   C77. An immunogenic composition comprising:        -   a. a liquid composition comprising (i) a first lipidated            polypeptide comprising the amino acid sequence set forth in            SEQ ID NO: 1; and (ii) a second lipidated polypeptide            comprising the amino acid sequence set forth in SEQ ID NO:            2; and        -   b. a lyophilized composition comprising            -   i. a Neisseria meningitidis serogroup A (MenA) capsular                saccharide conjugated to an adipic acid dihydrazide                (ADH) linker by 1-cyano-4-dimethylamino pyridinium                tetrafluoroborate chemistry, wherein the linker is                conjugated to tetanus toxoid carrier protein (TT) by                carbodiimide chemistry (MenA_(AH)-TT conjugate);            -   ii. a Neisseria meningitidis serogroup C (MenC) capsular                saccharide conjugated to an ADH linker by                1-cyano-4-dimethylamino pyridinium tetrafluoroborate                chemistry, wherein the linker is conjugated to tetanus                toxoid carrier protein (TT) by carbodiimide chemistry                (MenC_(AH)-TT conjugate);            -   iii. a Neisseria meningitidis serogroup W₁₃₅ (MenW)                capsular saccharide directly conjugated to tetanus                toxoid carrier protein (TT) by 1-cyano-4-dimethylamino                pyridinium tetrafluoroborate chemistry, in the absence                of a linker (MenW-TT conjugate); and            -   iv. a Neisseria meningitidis serogroup Y (MenY) capsular                saccharide directly conjugated to tetanus toxoid carrier                protein (TT) by 1-cyano-4-dimethylamino pyridinium                tetrafluoroborate chemistry, in the absence of a linker                (MenY-TT conjugate)                -   wherein the lyophilized composition is reconstituted                    with the liquid composition to produce the                    immunogenic composition.    -   C78. The immunogenic composition according to clause C77,        wherein the liquid composition further comprises aluminum.    -   C79. The immunogenic composition according to clause C77,        wherein the liquid composition further comprises aluminum        phosphate.    -   C80. The immunogenic composition according to clause C77,        wherein the lyophilized composition further comprises sodium        chloride.    -   C81. The immunogenic composition according to clause C77,        wherein the immunogenic composition comprises at least 0.010 mg        polysorbate-80 and at most 0.018 mg polysorbate-80.    -   C82. The immunogenic composition according to clause C77,        wherein the immunogenic composition comprises at least 0.01 mg        polysorbate-80 and at most 0.02 mg polysorbate-80.    -   C83. The immunogenic composition according to clause C77,        wherein the lyophilized composition does not contain aluminum.    -   C84. The immunogenic composition according to clause C78,        wherein the first polypeptide and the second polypeptide are        bound to the aluminum.    -   C85. The immunogenic composition according to clause C78,        wherein the first polypeptide and the second polypeptide are        bound to the aluminum in the immunogenic composition.    -   C86. The immunogenic composition according to clause C78,        wherein the concentration of polypeptides bound to the aluminum        in the immunogenic composition is not decreased after 24 hours,        as compared to the concentration of polypeptides bound to the        aluminum in the liquid composition prior to reconstituting the        lyophilized composition.    -   C87. The immunogenic composition according to clause C77,        wherein the concentration of MenA_(AH)-TT conjugate in the        immunogenic composition is not decreased after 24 hours, as        compared to the concentration of the MenA_(AH)-TT conjugate in        the lyophilized composition.    -   C88. The immunogenic composition according to clause C77,        wherein the concentration of MenC_(AH)-TT conjugate in the        immunogenic composition is not decreased after 24 hours, as        compared to the concentration of the MenCA_(H)-TT conjugate in        the lyophilized composition.    -   C89. The immunogenic composition according to clause C77,        wherein the concentration of MenW-TT conjugate in the        immunogenic composition is not decreased after 24 hours, as        compared to the concentration of the MenW-TT conjugate in the        lyophilized composition.    -   C90. The immunogenic composition according to clause C77,        wherein the concentration of MenY-TT conjugate in the        immunogenic composition is not decreased after 24 hours, as        compared to the concentration of the MenY-TT conjugate in the        lyophilized composition.    -   C91. The immunogenic composition according to clause C86,        wherein the concentration is decreased by at most 1% after 24        hours, as compared to the respective concentration in the liquid        composition prior to reconsititution.    -   C92. The immunogenic composition according to clause C86,        wherein the concentration is decreased by at most 5% after 24        hours, as compared to the respective concentration in the liquid        composition prior to reconsititution.    -   C93. The immunogenic composition according to clause C86,        wherein the concentration is decreased by at most 10% after 24        hours, as compared to the respective concentration in the liquid        composition prior to reconsititution.    -   C94. The immunogenic composition according to clause C87-C90,        wherein the concentration is decreased by at most 1% after 24        hours, as compared to the respective concentration in the        lyophilized composition prior to reconsititution.    -   C95. The immunogenic composition according to clause C87-C90,        wherein the concentration is decreased by at most 5% after 24        hours, as compared to the respective concentration in the        lyophilized composition prior to reconsititution.    -   C96. The immunogenic composition according to clause C87-C90,        wherein the concentration is decreased by at most 10% after 24        hours, as compared to the respective concentration in the        lyophilized composition prior to reconsititution.    -   C97. The immunogenic composition according to clause C68 or        clause C77, wherein the pH of the reconstituted immunogenic        composition is less than the pH of the lyophilized composition,        when reconstituted with sodium chloride.    -   C98. A composition comprising a) a first lipidated polypeptide        comprising the amino acid sequence set forth in SEQ ID NO: 1,        and b) a second lipidated polypeptide comprising the amino acid        sequence set forth in SEQ ID NO: 2.    -   C99. The composition according to clause C98, wherein the        composition further comprises polysorbate-80, aluminum,        histidine, and sodium chloride.    -   C100. The composition according to clause C98, wherein the        composition comprises 60 μg of the first lipidated polypeptide        and 60 μg of the second lipidated polypeptide.    -   C101. A method of inducing a bactericidal immune response        against a Neisseria meningitidis serogroup B subfamily A strain        and against a Neisseria meningitidis serogroup B subfamily B        strain in human, comprising administering to the human an        effective amount of the composition according to clause C1.    -   C102. A method of inducing a bactericidal immune response        against a Neisseria meningitidis serogroup B subfamily A strain        and against a Neisseria meningitidis serogroup B subfamily B        strain in human, comprising administering to the human an        effective amount of the composition according to clause C68.    -   C103. A method of inducing a bactericidal immune response        against a Neisseria meningitidis serogroup B subfamily A strain        and against a Neisseria meningitidis serogroup B subfamily B        strain in human, comprising administering to the human an        effective amount of the composition according to clause C77.    -   C104. A method of inducing a bactericidal immune response        against a Neisseria meningitidis serogroup A, a Neisseria        meningitidis serogroup C, a Neisseria meningitidis serogroup W,        and/or a Neisseria meningitidis serogroup Y strain in a human,        comprising administering to the human an effective amount of the        composition according to clause C1.    -   C105. A method of inducing a bactericidal immune response        against a Neisseria meningitidis serogroup A, a Neisseria        meningitidis serogroup C, a Neisseria meningitidis serogroup W,        and/or a Neisseria meningitidis serogroup Y strain in a human,        comprising administering to the human an effective amount of the        composition according to clause C68.    -   C106. A method of inducing a bactericidal immune response        against a Neisseria meningitidis serogroup A, a Neisseria        meningitidis serogroup C, a Neisseria meningitidis serogroup W,        and/or a Neisseria meningitidis serogroup Y strain in a human,        comprising administering to the human an effective amount of the        composition according to clause C77.    -   C107. A method of inducing a bactericidal immune response        against a Neisseria meningitidis serogroup A, Neisseria        meningitidis serogroup B, a Neisseria meningitidis serogroup C,        a Neisseria meningitidis serogroup W, and/or a Neisseria        meningitidis serogroup Y strain in a human, comprising        administering to the human an effective amount of the        composition according to clause C1.    -   C108. A method of inducing a bactericidal immune response        against a Neisseria meningitidis serogroup A, Neisseria        meningitidis serogroup B, a Neisseria meningitidis serogroup C,        a Neisseria meningitidis serogroup W, and/or a Neisseria        meningitidis serogroup Y strain in a human, comprising        administering to the human an effective amount of the        composition according to clause C68.    -   C109. A method of inducing a bactericidal immune response        against a Neisseria meningitidis serogroup A, Neisseria        meningitidis serogroup B, a Neisseria meningitidis serogroup C,        a Neisseria meningitidis serogroup W, and/or a Neisseria        meningitidis serogroup Y strain in a human, comprising        administering to the human an effective amount of the        composition according to clause C77.    -   C110. A method of inducing a bactericidal immune response        against a Neisseria meningitidis serogroup A, Neisseria        meningitidis serogroup B, a Neisseria meningitidis serogroup C,        a Neisseria meningitidis serogroup W, a Neisseria meningitidis        serogroup Y strain, and a Neisseria meningitidis serogroup X        strain in a human, comprising administering to the human an        effective amount of the composition according to clause C1.    -   C111. A method of inducing a bactericidal immune response        against a Neisseria meningitidis serogroup A, Neisseria        meningitidis serogroup B, a Neisseria meningitidis serogroup C,        a Neisseria meningitidis serogroup W, a Neisseria meningitidis        serogroup Y strain, and a Neisseria meningitidis serogroup X        strain in a human, comprising administering to the human an        effective amount of the composition according to clause C68.    -   C112. A method of inducing a bactericidal immune response        against a Neisseria meningitidis serogroup A, Neisseria        meningitidis serogroup B, a Neisseria meningitidis serogroup C,        a Neisseria meningitidis serogroup W, a Neisseria meningitidis        serogroup Y strain, and a Neisseria meningitidis serogroup X        strain in a human, comprising administering to the human an        effective amount of the composition according to clause C77.    -   C113. A method of inducing a bactericidal immune response        against a Neisseria meningitidis serogroup A, Neisseria        meningitidis serogroup B, a Neisseria meningitidis serogroup C,        a Neisseria meningitidis serogroup W, a Neisseria meningitidis        serogroup Y strain, and a Neisseria meningitidis serogroup X        strain in a human, comprising administering to the human an        effective amount of the composition according to clause C98.    -   C114. A method of inducing a bactericidal immune response        against N. meningitidis serogroup C strain expressing factor H        binding protein A10 in a human, the method comprising        administering to the human a composition comprising a first        lipidated polypeptide comprising the amino acid sequence set        forth in SEQ ID NO: 1; a second lipidated polypeptide comprising        the amino acid sequence set forth in SEQ ID NO: 2.    -   C115. A method of inducing a bactericidal immune response        against N. meningitidis serogroup W strain expressing factor H        binding protein A10 in a human, comprising administering to the        human a composition comprising a polypeptide comprising the        amino acid sequence set forth in SEQ ID NO: 1.    -   C116. A method of inducing a bactericidal immune response        against N. meningitidis serogroup W strain expressing factor H        binding protein A19 in a human, comprising administering to the        human a composition comprising a polypeptide comprising the        amino acid sequence set forth in SEQ ID NO: 1.    -   C117. A method of inducing a bactericidal immune response        against N. meningitidis serogroup A strain expressing factor H        binding protein B16 in a human, comprising administering to the        human a composition comprising a polypeptide comprising the        amino acid sequence set forth in SEQ ID NO: 2.    -   C118. A method of inducing a bactericidal immune response        against N. meningitidis serogroup Y strain expressing factor H        binding protein B47 in a human, comprising administering to the        human a composition comprising a polypeptide comprising the        amino acid sequence set forth in SEQ ID NO: 2.    -   C119. A method of inducing a bactericidal immune response        against N. meningitidis serogroup C strain expressing factor H        binding protein A10 in a human, the method comprising        administering to the human a composition comprising a first        lipidated polypeptide comprising the amino acid sequence set        forth in SEQ ID NO: 1; a second lipidated polypeptide comprising        the amino acid sequence set forth in SEQ ID NO: 2.    -   C120. A method of inducing a bactericidal immune response        against N. meningitidis serogroup W strain expressing factor H        binding protein A10 in a human, comprising administering to the        human a composition comprising a first lipidated polypeptide        comprising the amino acid sequence set forth in SEQ ID NO: 1; a        second lipidated polypeptide comprising the amino acid sequence        set forth in SEQ ID NO: 2.    -   C121. A method of inducing a bactericidal immune response        against N. meningitidis serogroup W strain expressing factor H        binding protein A19 in a human, comprising administering to the        human a composition comprising a first lipidated polypeptide        comprising the amino acid sequence set forth in SEQ ID NO: 1; a        second lipidated polypeptide comprising the amino acid sequence        set forth in SEQ ID NO: 2.    -   C122. A method of inducing a bactericidal immune response        against N. meningitidis serogroup A strain expressing factor H        binding protein B16 in a human, comprising administering to the        human a composition comprising a first lipidated polypeptide        comprising the amino acid sequence set forth in SEQ ID NO: 1; a        second lipidated polypeptide comprising the amino acid sequence        set forth in SEQ ID NO: 2.    -   C123. A method of inducing a bactericidal immune response        against N. meningitidis serogroup Y strain expressing factor H        binding protein B47 in a human, comprising administering to the        human a composition comprising a first lipidated polypeptide        comprising the amino acid sequence set forth in SEQ ID NO: 1; a        second lipidated polypeptide comprising the amino acid sequence        set forth in SEQ ID NO: 2    -   C124. A method of inducing a bactericidal immune response        against a Neisseria meningitidis serogroup X strain in a human,        comprising administering to the human an effective amount of a        polypeptide having at least 70% identity to the amino acid        sequence selected from the group consisting of SEQ ID NO: 1, SEQ        ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:        9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13,        SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ        ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID        NO: 22, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO:        28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32,        SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ        ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID        NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO:        45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49,        SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ        ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID        NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID        NO: 62.    -   C125. A method of inducing a bactericidal immune response        against a Neisseria meningitidis serogroup X strain in a human,        comprising administering to the human an effective amount of the        composition according to clause C1.    -   C126. A method of inducing a bactericidal immune response        against a Neisseria meningitidis serogroup X strain in a human,        comprising administering to the human an effective amount of the        composition according to clause C68.    -   C127. A method of inducing a bactericidal immune response        against a Neisseria meningitidis serogroup X strain in a human,        comprising administering to the human an effective amount of the        composition according to clause C77.    -   C128. A method of inducing a bactericidal immune response        against a Neisseria meningitidis serogroup X strain in a human,        comprising administering to the human an effective amount of the        composition according to clause C98.    -   C129. A method of inducing a bactericidal immune response        against N. meningitidis serogroup X strain expressing factor H        binding protein B49 in a human, comprising administering to the        human a composition according to clause C98.    -   C130. A method of inducing a bactericidal immune response        against N. meningitidis serogroup X strain expressing factor H        binding protein B49 in a human, comprising administering to the        human a composition comprising a first lipidated polypeptide        comprising the amino acid sequence set forth in SEQ ID NO: 1; a        second lipidated polypeptide comprising the amino acid sequence        set forth in SEQ ID NO: 2.    -   C131. The method according to any one of clauses C101-C130,        wherein the composition induces a bactericidal titer of serum        immunoglobulin that is at least 2-fold higher in the human after        receiving the first dose than a bactericidal titer of serum        immunoglobulin in the human prior to receiving the first dose,        when measured under identical conditions in a serum bactericidal        assay using human complement.    -   C132. The method according to any one of clauses C101-C130,        wherein the composition induces a bactericidal titer of serum        immunoglobulin that is at least 4-fold higher in the human after        receiving the first dose than a bactericidal titer of serum        immunoglobulin in the human prior to receiving the first dose,        when measured under identical conditions in a serum bactericidal        assay using human complement.    -   C133. The method according to any one of clauses C101-C132,        wherein the composition induces a bactericidal titer of serum        immunoglobulin that is at least 8-fold higher in the human after        receiving the first dose than a bactericidal titer of serum        immunoglobulin in the human prior to receiving the first dose,        when measured under identical conditions in a serum bactericidal        assay using human complement.    -   C134. The method according to any one of clauses C101-C132,        wherein the patient is aged 12 to <18 Months or 18 to <24        Months.    -   C135. The method according to any one of clauses C101-C132,        wherein the patient is aged 18 to <24 Months.    -   C136. The method according to any one of clauses C101-C132,        wherein the patient is aged ≥24 Months to <10 Years.    -   C137. A method for eliciting an immune response in a patient,        comprising: (a) administering an immunogenic composition to the        patient when the patient is aged between 12 and 18 months;        wherein the immunogenic composition comprises a first lipidated        polypeptide comprising the amino acid sequence set forth in SEQ        ID NO: 1; and a second lipidated polypeptide comprising the        amino acid sequence set forth in SEQ ID NO: 2.    -   C138. A method for eliciting an immune response in a patient,        comprising: (a) administering an immunogenic composition to the        patient when the patient is aged between 18 and 24 months;        wherein the immunogenic composition comprises a first lipidated        polypeptide comprising the amino acid sequence set forth in SEQ        ID NO: 1; and a second lipidated polypeptide comprising the        amino acid sequence set forth in SEQ ID NO: 2.    -   C139. A method for eliciting an immune response in a patient,        comprising: (a) administering an immunogenic composition to the        patient when the patient is aged between 24 months and 10 years;        wherein the immunogenic composition comprises a first lipidated        polypeptide comprising the amino acid sequence set forth in SEQ        ID NO: 1; and a second lipidated polypeptide comprising the        amino acid sequence set forth in SEQ ID NO: 2.    -   C140. The method according to any one of clauses C137-C139,        wherein the composition further comprises polysorbate-80,        aluminum, histidine, and sodium chloride.

What is claimed is:
 1. A method of inducing an immune response against aNeisseria meningitidis serogroup A strain expressing B16 factor Hbinding protein in a human comprising mixing (a) a compositioncomprising (i) a first lipidated polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 1; (ii) a second lipidated polypeptidecomprising the amino acid sequence set forth in SEQ ID NO: 2; (iii) aNeisseria meningitidis serogroup A (MenA) capsular saccharide conjugatedto an adipic acid dihydrazide (ADH) linker, wherein the linker isconjugated to tetanus toxoid (TT); (iv) a Neisseria meningitidisserogroup C (MenC) capsular saccharide conjugated to an ADH linker,wherein the linker is conjugated to tetanus toxoid (TT); (v) a Neisseriameningitidis serogroup W135 (MenW) capsular saccharide conjugated totetanus toxoid (TT) (MenW-TT conjugate); and (vi) a Neisseriameningitidis serogroup Y (MenY) capsular saccharide directly conjugatedto tetanus toxoid (TT) (MenY-TT conjugate); and (b) administering aneffective amount of the composition to the human, wherein thecomposition further comprises aluminum.
 2. The method according to claim1, wherein at least 90% of the total composition of the firstpolypeptide is bound to aluminum in the composition.
 3. The methodaccording to claim 1, wherein at least 90% of the total composition ofthe second polypeptide is bound to aluminum in the composition.
 4. Themethod according to claim 1, wherein the composition further comprisespolysorbate-80.
 5. The method according to claim 1, wherein thecomposition further comprises sucrose.
 6. The method according to claim1, wherein the composition further comprises histine.
 7. The methodaccording to claim 1, wherein the composition comprises about 120 μg/mlof the first polypeptide; about 120 μg/ml of the second polypeptide;about 0.5 mg/ml aluminum as aluminum phosphate; about 0.02 mgpolysorbate-80; about 10 mM histidine; and about 150 mM sodium chloride.8. The method according to claim 1, wherein the composition comprisesabout 60 μg of the first polypeptide; about 60 μg of the secondpolypeptide; about 5 μg of the MenA capsular saccharide conjugated toabout 7.5 μg TT; about 5 μg of the MenC capsular saccharide conjugatedto about 7.5 μg TT; about 5 μg of the MenW capsular saccharideconjugated to about 3.75 μg TT; about 5 μg of the MenY capsularsaccharide conjugated to about 3.25 μg TT; about 97 μg Tris-HCl, pH6.80.3; 4.69-4.71 mg of sodium chloride; about 28 mg of sucrose; about0.78 mg of L-Histidine; about 0.02 mg polysorbate-80; about 0.25 mgaluminum; and further comprising 0.5 mL water, per dose.
 9. The methodaccording to claim 1, wherein the first polypeptide consists of theamino acid sequence set forth in SEQ ID NO:
 1. 10. The method accordingto claim 1, wherein the second polypeptide consists of the amino acidsequence set forth in SEQ ID NO:
 2. 11. The method according to claim 1,wherein the composition does not comprise a hybrid protein.
 12. Themethod according to claim 1, wherein the composition does not comprise afusion protein.
 13. The method according to claim 1, wherein thecomposition is not lyophilized.
 14. The method according to claim 1,wherein the composition does not comprise formaldehyde.
 15. The methodaccording to claim 1, wherein the composition does not comprisediphtheria toxoid or CRM.
 16. The method according to claim 1, whereinthe human is aged less than 12 months.
 17. The method according to claim1, wherein the human is aged 12 months to 18 months.
 18. The methodaccording to claim 1, wherein the human is aged 18 months to 24 months.19. The method according to claim 1, wherein the human is aged 24 monthsto 10 years.
 20. The method according to claim 1, wherein the human isaged at least 10 years.
 21. The method according to claim 1, wherein thehuman is aged 10 to 25 years.